Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
Read-across from supporting substance (structural analogue or surrogate): In 13-week dermal subchronic toxicity studies in rats and mice with sodium thioglycolate, no treatment-related effects on sperm density and motility, caudal epididymal sperm, spermatid head counts in the testes and testis weights, as well as oestrous cycles, were observed up to dose levels of 180 and 360 mg/kg bw/d, respectively. 
Link to relevant study records

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Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 December 2005 - 14 September 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
ANIMALS
- Breeder: Charles River Laboratories France, L'Arbresle, France
- Age at the beginning of the treatment period: 6 weeks old 
- Weight at the beginning of the treatment period: ca. 205 g for the  males and ca. 160 g for the females
- Acclimation: 7-days before the beginning of the treatment period

ENVIRONMENTAL CONDITIONS
- Temperature : 22 ± 2°C
- Relative humidity : 50 ± 50%
- Light/dark cycle : 12h/12h (7:00 - 19:00)
- Ventilation : about 12 cycles/hour of filtered, non-recycled air.

HOUSING
The animals were housed individually in polycarbonate cages or in  wire-wesh cages. Autoclaved wood shavings were provided as nesting  material, a few days before delivery and during the lactation period

FOOD and WATER
- Food: SSNIFF R/M-H pelleted maintenance diet ad libitum
- Water: filtered (0.22 µm filter) tap water ad libitum
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
TREATMENT
- Vehicle: degassed purified water, obtained by reverse osmosis 
- Dosage form preparation: solution in the vehicle at 4, 8 and 16 mg/mL,  expressed as active substance. 
- Volume: 5 ml/kg
Details on mating procedure:
MATING
- Mating procedure: one female was placed with one male from the same  dose-level group. If necessary, the estrous cycle stage was determined  from a fresh vaginal lavage (stained with methylene blue), each morning  during the mating period, until the females mated

PARTURITION
Females were allowed to drop their litters normally and rear their  progeny until sacrifice
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On weeks 1, 4, 8 and 12. There was a satisfactory agreement (± 10%) between the nominal and  actual concentrations.
Duration of treatment / exposure:
Exposure period: * Males: during 10w before mating, the mating period (2w) and until sacrifice.
* Females: during 10 w before mating, the mating period, pregnancy and lactation until day 4 post partum.
Premating exposure period (males): 10 weeks
Premating exposure period (females): 10 weeks
Duration of test: 16 weeks
Frequency of treatment:
7 days per week
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
80 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected by the Study Monitor based on the results of a preliminary 2-week toxicity study (CIT/Study No. 30720 TSR). In this study sodium thioglycolate in purified water was planned to be administered at dose-levels of 15, 30, 60 and 75 mg/kg/dayfor 14 days. After no effects were observed at all dose-levels, the group previously given 15 mg/kg/day was administered with 100 mg/kg/day on days 8, 9 and 10 and then 150 mg/kg/day on days 11, 12 and 13, the other groups were treated as scheduled until day 14. When treated at 150 mg/kg/day, one male was sacrificed on day 13 following marked clinical signs and three females were found dead on day 14. The male and two of the females had spleens reduced in size. Male terminal body weight gains were slightly lower than controls at 15/100/150 mg/kg/day while females showed a body weight gain comparable to controls until day 11 and a mean terminal body weight loss (-10%) when the dose level was increased to 150 mg/kg/day. Food consumption was lower for this group for both sexes over the last 3-day period when the dose level was increased to 150 mg/kg/d. At necropsy, some animals in the 60, 75 and 15/100/150 mg/kg/day dose-groups had irregular colored kidneys, while at 75 mg/kg/day marked lobular pattern of the liver was noted with paleness (also seen at 15/100/150 mg/kg/day). Mean ovary weights were lower at 15/100/150 mg/kg/day and mean uterus weights were lower at 75 and 15/100/150 mg/kg/day. 150 mg/kg/day was considered to exceed the maximum tolerated dose because mortality occurred after 3 days of treatment. 80 mg/kg/day was selected as a top dose level for the OECD 421 study considering the limited effects observed at 75 mg/kg/day.
Positive control:
No apprpriate
Parental animals: Observations and examinations:
- Morbidity and mortality: at least twice a day
- Clinical signs: at least once a day
- Body weight:          
. males: on day 1, then once a week until sacrifice         
. females: on day 1, then once a week until mated, then on days 0, 7, 14  and 20 pc and on days 1 and 5 pp (postpartum).
- Food consumption         
. males: once a week (except during the mating period) until sacrifice         
. females: once a week during the premating period and then on the  following intervals: days 0-7, 7-10, 10-14, 14-17 and 17-20 pc, and days  1-5 pp.
Estrous cyclicity (parental animals):
Each morning for three weeks before the start of the pairing period. 
Sperm parameters (parental animals):
The left epididymis was removed, weighed (total and cauda separately) and  sperm from the cauda was sampled for motility, morphology investigations  and epididymal sperm count. 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) was counted in a Neubauer cell.
Litter observations:
- Litter size: total litter size and numbers of pups of each sex were  recorded as soon as possible after birth. The litters were observed daily.
- Clinical signs: daily
- Body weight: days 1 and 5 pp
Postmortem examinations (parental animals):
PATHOLOGY
- Sacrifice
. males: after the end of the mating period,
. females: on day 5 pp,
. females which had not delivered by day 25 pc: on day 25 pc
. females which did not mate: 24 days after the end of the mating period,
- Organ weights: 
Brain, epididymides, heart, kidneys, liver, ovaries,  prostate, seminal vesicles and testes, uterus
- Macroscopic post-mortem examination: on all parent animals. In all  females, the number of implantation sites and corpora lutea were  recorded. 
- Preservation of tissues: macroscopic lesions, ovaries, prostate,  seminal vesicles, uterus (horns and cervix), vagina, brain, heart, liver  and kidneys, in 10% buffered formalin. Testes and epididymides, in  Bouin's fluid        
- Microscopic examination: macroscopic lesions,  epididymides, heart,  kidneys, liver, ovaries, prostate*, seminal vesicles*, testes, and  uterus (* in the control and high-dose groups only).
Postmortem examinations (offspring):
PATHOLOGY
- Sacrifice
. pups: on day 5 pp.
-  A macroscopic examination was performed for all pups, including those found dead or prematurely sacrificed. Macroscopic lesions were preserved in 10% buffered formalin (or another appropriate fixative).
Statistics:
- Data other than organ weights:
Mean values were compared by one-way variance analysis and Dunnett test, (mean values being considered as normally distributed, variances being considered as homogeneous). Values compared by Fisher exact probability test are presented as percentages.
- Organ weights:
Dunn test or Student test (2 groups) or Dunnett test (3 or more groups) or Mann-Whitney/Wilcoxon test
Reproductive indices:
Pre-implantation loss:
Number of corpora lutea - Number of implantation sites
_____________________________________________ x 100
Number of corpora lutea

Post-implantation loss:
Number of implantation sites - Number of live concepti
_____________________________________________ x 100
Number of implantations

Mating index:
Number of mated animals
_____________________ x 100
Number of paired animals

Fertility index:
Number of pregnant female partners
_______________________________ x 100
Number of mated pairs

Gestation index:
Number of females with live born pups
________________________________ x 100
Number of pregnant females
Offspring viability indices:
Live birth index:
Number of live born pups
_____________________ x 100
Number of delivered pups

Viability index on day 4 post-partum:
Number of surviving pups on day 4 post-partum
_______________________________________ x 100
Number of live born pups
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see below
Mortality:
mortality observed, treatment-related
Description (incidence):
see below
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see below
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: estrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
see below
CLINICAL EXAMINATIONS
- Mortality: 
. 80 mg/kg/day:
One female (K20428) was found dead on day 23 of dosing. No clinical signs other than ptyalism had been observed prior to death. No abnormalities were observed at post-mortem examination.
One male (K20347) was found dead on day 74 of dosing. No clinical signs other than ptyalism were observed prior to death. The male had not mated prior to death. At necropsy, the stomach mucosa had brown/black areas.
One male (K20349) was found dead on day 90 of dosing. No clinical signs other than ptyalism were observed prior to death. At necropsy, the right kidney had a dilated pelvis.
One female (K20421) was found dead on day 23 post-coitum with 17 live pups in the bedding. At necropsy, thick, blackish contents were observed in the vagina.
One female (K20422) was found dead on day 23 post-coitum with 4 dead pups and one live pup in the bedding and one pup in the vagina. At necropsy, there were 11 dead fetuses in the uterine horns.
One female (K20426) was found dead on day 23 post-coitum with 11 live pups and five dead pups in the bedding. Difficulty to deliver was noted for the female prior to death. At necropsy, the lungs were reddish in color and there were serous contents in the thoracic cavity.
One female (K20427) was prematurely sacrificed on day 23 post-coitum with signs of piloerection, cold to the touch, pallor of eye recorded only on the same day. No pups were delivered prior to death. At necropsy, there were 15 dead fetuses and one live fetus and one late resorption in the uterine horns and the liver was pale.
One female (K20429) was prematurely sacrificed on day 1 post-partum because all the pups were dead. Prior to sacrifice, emaciated appearance, piloerection and round back were observed. No abnormalities were observed at post-mortem examination.
One female (K20423) was found dead on day 2 post-partum with no clinical signs other than ptyalism observed prior to death. At necropsy, the urinary bladder was dilated.
. 40 mg/kg/day:
One female (K20415) was found dead on day 22 post-coitum. At necropsy, the female was pregnant with 16 dead fetuses in the uterine horns.
The high rate of mortality in the treated females, with presence of implantation scars, late resorptions and/or dead fetuses in the uterine horns, was considered to be treatment-related.

- Clinical symptoms: 
All males and females given 80 mg/kg/day were observed to have ptyalism from week 2 of dosing generally until the end of the study. The number of females affected during gestation (8/11) and lactation (2/4) was less than the number affected during pre-pairing (11/12).
7/12 males and 5/12 females given 40 mg/kg/day experienced ptyalism towards the end of the pre pairing dosing period (week 9 onwards) and, for males, until the end of the study, but only 3/12 females were affected during gestation and 1/11 had ptyalism during lactation.
No clinical signs were observed at 20 mg/kg/day.
- Body weight: Table 1
The mean body weight gain over the 10-week pre-mating period was similar to that of the controls for both males and females at all dose-levels. Females given 80 mg/kg/day had a slight lowering of body weight gain between day 50 and day 64 but this did not affect the overall mean gain.
There were no effects of treatment on body weight gains during gestation. Females treated at all dose-levels showed an apparently increased body weight gain during the lactation period, however there were two control females which lost weight during this period and when they are excluded there are no significant differences between the groups (there were only two females in the group mean at 80 mg/kg/day so the mean value is not as reliable as the other groups).
- Food consumption: 
Male food consumption at all dose-levels was similar to that of the controls during the first 5 weeks of dosing (ranging from -3% to +4% difference) and was then statistically significantly higher at 80 mg/kg/day for the last 5 weeks of the pre mating period (+10% to +14%).
Female food consumption at all dose-levels was similar to that of the controls throughout the pre mating and gestation periods (ranging from -5% to +16% difference). Females given 80 mg/kg/day had slightly lower mean food consumption during lactation (-10%), but given the low number of females considered in this dose group, these changes were considered to bear no biological significance.

MATING AND FERTILITY DATA
- Oestrus cycle: 
There were no effects of treatment with sodium thioglycolate on vaginal cyclicity, with mean cycle lengths of 4 to 5 days in all groups including control.
- Mating data:
With the exception of male K20347 (given 80 mg/kg/day) which was found dead after 3 days of pairing and had not mated, all pairs mated. The mean number of days taken to mate was slightly higher for the group given 40 mg/kg/day but as this was related to the contribution of two females and as it was not also observed at 80 mg/kg/day, it was considered not to be related to treatment.
- Fertility data:
There was one non-pregnant female in each of the groups given vehicle or 20 mg/kg/day and two non-pregnant females in the group given 80 mg/kg/day. This was considered not to be related to treatment with the test item.
- Delivery data: Table 2
The duration of gestation was statistically significantly longer for the females given 80 mg/kg/day than for the other groups and the number of females surviving delivery was markedly lower.
The mean number of corpora lutea was lower in females given 80 mg/kg/day resulting in a lower number of implantations and fetuses, although implantation losses were comparable with the controls and observed only in one female with total resorptions.
Pup survival was slightly worse at 80 mg/kg/day, mainly due to one female found dead so her litter was sacrificed and one female with a dead litter. The remaining two females lost only three pups between them.

PATHOLOGY
- Semiology: 
More than 96% of the sperm were morphologically normal in the groups given SODIUM THIOGLYCOLATE and more than 95% of the sperm were motile. There were therefore no effects of treatment at any dose-level.
All values fore epididymidal and testicular sperm counts are similar to, or greater than, those observed in controls, there were therefore no effects of treatment.
- Organ weights: Table 3
Higher statistically significant absolute and relative kidney and liver weights were noted in the males given 80 mg/kg/day compared with their respective controls. Higher liver weights correlated in the males given 80 mg/kg/day with a trend in higher glycogen content. The increased liver weights were considered to be related to the test item administration in view of the effect of SODIUM THIOGLYCOLATE on the carbohydrate metabolism. For the higher kidney weights, a relationship to treatment was considered to be equivocal as there were no histopathological correlates.
Dose-related lower absolute and relative seminal vesicle weights (statistically significant for the absolute weights at 20 and 40 mg/kg/day and for the absolute and relative weights at 80 mg/kg/day) were noted in the treated males. The seminal vesicle weights of some control males were higher than CIT Sprague-Dawley historical control data range (mean: 1,618/0.320; minimum: 1,185/0.257; maximum: 1,958/0,461 in absolute and relative weights repectively). Consequently, the lower seminal vesicle weights of the males given 20 or 40 mg/kg/day were not considered to be toxicologically relevant while they were considered to be treatment-related at 80 mg/kg/day, since they correlated with slight decrease in secretory content in the seminal vesicles.
Slightly lower absolute and relative prostate weights were noted in the males given 80 mg/kg/day. This was the contribution of 3/10 animals with individual prostate weights below the control range. No correlates were noted at microscopic examination of group 4 and a relationship to treatment was accordingly considered to be unlikely.
Higher mean absolute and relative uterus weights noted in the females given 80 mg/kg/day was the contribution of two females among the five surviving females. The particularly high uterus weight was due to green contents or black content together with late resorption, respectively.
- Macroscopic post-mortem examination: 
. Animals prematurely killed or found dead
Males
No major factors contributing to death were observed in the two males found dead at 80 mg/kg/day. Brown/black areas noted in stomach mucosa and right kidney dilated pelvis noted respectively in male numbers K20347 and K20349 were considered to be without relationship to treatment with the test item.
Females
One among 12 females given 40 mg/kg/day and 7/12 females given 80 mg/kg/day were found dead or prematurely sacrificed during pregnancy or lactation.
For females K20423 and K20428 (80 mg/kg/day) found dead at day 98 (day 2 post-partum) or 23, respectively and for female K20429 (80 mg/kg/day) prematurely sacrificed at day 101 (day 1 post partum), no necropsy observations were recorded, except dilated urinary bladder for female K20423. For almost all other females found dead or prematurely killed macroscopic observations were noted in the uterine horns and sometimes in vagina.
In addition, the liver was pale in female K20427 (80 mg/kg/day) prematurely killed on day 98 (day 23 post-coitum). This correlated with marked vacuolated foamy hepatocytes and minimal hepatocellular degeneration/necrosis and granulocyte infiltration. A relationship to treatment was considered to be unlikely as such finding was not found in the females which survived nor in the males.
. Survivors
Males
Seminal vesicles were of small size in 1/12 males given 80 mg/kg/day. This lower weight correlated with reduced seminal vesicle secretion and was considered to be treatment-related.
Females
Uterine horn content and/or vaginal content in females killed at termination

Dose-level (mg/kg/day) 0 20 40 80
Liquid green content/dilatation (horns) - - - (K0) K20425
Late Resorption (left horn) - - - (K0) K20431
Thick black content (left horn) - - - (K0) K20431
Thick black content (vagina) - - - (K0) K20431
(K0): scheduled sacrifice.
Late resorption was considered to be treatment-related.

- Microscopic examination:         
. Males
Systemic (scheduled sacrifices and unscheduled sacrifices)
* Heart
Minimal or slight myocardial degeneration/necrosis was seen in the two males given 80 mg/kg/day and found dead at day 74 or 90.
As these findings were recorded focally and moreover also found in the control animals which survived (2/12 males), they were considered without relationship to treatment. This finding is commonly seen in untreated Sprague-Dawley rats of this age (Greaves, 2007).
* Liver
A trend in marginally higher concentration of glycogen content was observed in the liver of the surviving males given 80 mg/kg/day.
Incidence and mean severity [affected and (affected + non affected)] of glycogen content in the liver of decedents and surviving males

Dose-level (mg/kg/day) 0 20 40 80
Number of males with glycogen content
Status: found dead - - - 0/2
Number of males with glycogen content
Status: scheduled sacrifice 10/12 10/12 8/12 9/10
Mean severity (affected animals) (3) (2.5) (2.1) (3.9)
Mean severity (affected + non affected) 2.5 2.1 1.4 3.5
-: not applicable.

This finding was considered to be related to the test item administration in view of the effect of SODIUM THIOGLYCOLATE on the carbohydrate metabolism. However these marginal differences at microscopic examination of the liver given the high dose-level were considered not to be adverse.
In addition, a relationship to the slightly higher liver weights at 80 mg/kg/day in the males which survived and which were not fasted before sacrifice was considered to be uncertain as the marginally lower glycogen content at 40 mg/kg/day in the males was not correlated with lower liver weights.
* Kidneys
Acidophilic globules were noted in the cortical tubular epithelium of the kidneys of the males given 80 mg/kg/day and of their respective controls. Incidence and severity were similar in both groups. It was therefore considered not to be toxicologically meaningful and related to the intra tubular alpha micro-globulin deposits in renal cortex which can be found at such incidence and severity in treated as well as untreated control male rats.

Incidence and mean severity (affected and affected + non affected) of acidophilic globules in the kidneys of decedents and surviving males

Dose-level (mg/kg/day) 0 80
Number of males with acidophilic globules
(mean severity)
Status: found dead - 1/2 (2.0)
Number of males with acidophilic globules
(mean severity)
Status: scheduled sacrifice 9/12 (1.7) 8/10 (1.9)
Total number of males and mean severity 9/12 (1.7) 9/12 (1.9)
Mean severity (affected + non affected) 1.3 1.4
-: not applicable.

* Genital Organs
Testes/Epididymides
The microscopic examination of the PAS/Hematoxylin stained testes and epididymides with knowledge of the different stages of maturation of the seminiferous tubules did not reveal any disturbance in the treated males. There was no evidence of degenerative changes or delay in sexual maturity in the treated males from any group compared with the respective controls. The higher number of males given 40 mg/kg/day with focal tubular atrophy was considered to be fortuitous and correspond to the junction between seminiferous tubules and tubulu recti, as it was almost always found near albuginea (subcapsular).
Seminal vesicles
Slight reduced secretory content was noted in 5/10 surviving males given 80 mg/kg/day. It correlated with lower weights at necropsy and was considered to be treatment-related. Reduced secretory content, most often unilateral and of minimal or slight severity, was also observed in 5/12 males given 20 mg/kg/day. Unilateral reduced secretory content was noted at minimal severity in 3/12 males given 40 mg/kg/day, while unilateral slight or moderate reduced secretory content was observed in two other males given 40 mg/kg/day. Except for male K20328 given the low dose-level, the individual seminal vesicle weights of the males given 20 or 40 mg/kg/day were of same magnitude as those of the controls.
It was considered that the reduced secretory content at 20 and 40 mg/kg/day was not treatment related.
In addition, as no atrophy of seminal epithelium could be observed in any treated group, the reduced secretory content together with lower seminal vesicle weights at 80 mg/kg/day was considered not to be adverse. This was confirmed by the absence of any abnormality in the sperm parameters.

- Females
Systemic (scheduled sacrifices and unscheduled sacrifices)

* Heart
Minimal myocardial degeneration/fibrosis was seen in 1/7 females prematurely killed. As this finding was recorded focally, and moreover found in one control female that survived, it was not considered to be related to the test item administration.

* Liver
Slight or marked peri/medio-lobular vacuolated hepatocytes were seen in 3/7 females found dead or prematurely sacrificed given 80 mg/kg/day. In 1/7 females marked vacuolated hepatocytes were associated with minimal hepatocellular degeneration/necrosis and minimal granulocyte infiltration. A relationship to treatment was considered to be equivocal as such observations were not found in any surviving female given 80 mg/kg/day. In control and treated (80 mg/kg/day) groups, half of the females (almost all surviving and one found dead) showed similar severity of glycogen content (see table below).

Incidence and mean severity (affected and (affected + non affected)) of glycogen content in the liver of decedents and surviving females

Dose-level (mg/kg/day) Number of females with glycogen content
(mean severity)
0 80
Status: found dead - 1/7 (3.0)
Status: scheduled sacrifice 6/12 (2.3) 4/5 (2.8)
Total number of females (affected animals) 6/12 (2.3) 5/12 (2.8)
Mean severity (affected + non affected) 1.2 1.2
-: not applicable.

* Kidneys
Slight bilateral vacuolated cortical tubules, tubular dilatation and unilateral, minimal, focal cortical necrosis were observed in the kidneys of female K20427 given 80 mg/kg/day and sacrificed moribund. Slight proteinaceous casts were noted in the kidneys of female K20421 from the same group. None of these were noted in any of the surviving females. All were thus considered to be of no toxicological importance.

* Uterus/vagina
Microscopic examination of uterus and/or vagina was suggestive of on-going pregnancy in the females found dead or prematurely sacrificed or evidence that females had not delivered. It was recorded in the table 4.
No relevant microscopic observations were recorded in the ovaries.

All the other microscopic findings noted in the prematurely killed, found dead or surviving males and females given 80 mg/kg/day or from the control group, including those observed in the prostate of the males, were those which can be found spontaneously in the untreated laboratory rat of this strain and were considered to bear no relationship to treatment with the test item.

Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
mortality
Key result
Dose descriptor:
LOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
mortality
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
see below
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
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
Histopathological findings:
not examined
Other effects:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
OBSERVATION OF THE PUPS AFTER BIRTH
- Mortality: 
Pup survival was slightly worse at 80 mg/kg/day, mainly due  to one female was found dead so her litter was sacrificed and one female  had a dead litter. The remaining two females lost only two pups between  them.
- Clinical signs: 
One pup in the control group had a haematoma on the  head and one pup in the 40 mg/kg/day dose-group had a necrosed tail. As  these were isolated findings, they were considered to be spontaneous in  origin. 
- Pup body weight: 
Pups of dams treated at 40 or 80 mg/kg/day showed an increased mean body weight gain from day 1 to day 5 post-partum (ranging from +14% to +51% difference). The pups of dams treated at 80 mg/kg/day also had a higher mean birth weight than the controls (+12% for males and +14% for females) but there were fewer pups per litter in this group which is likely to have had an impact. Since this increased body weight gain is dose-related it is considered to be related to treatment with the test item.
- Sex ratio: 
The percentage of male pups was slightly low at 80 mg/kg/day  (ns), when compared with the controls, however there were less pups in  this group.
- Pup necropsy findings: 
One pup in the 40 mg/kg/day dose-group had a small liver with whitish areas. One pup in the 20 mg/kg/day group had whitish areas on the liver.
These findings were not observed in the 80 mg/kg/day dose-group. The examination of  the historical control data shown that whitish area have already been  observed in control animals in 2 OECD 421 studies with frequencies of  1/125 (0.8%) and 1/113 (0.9%), in consequence this effect was not  considered to be treatment-related.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
viability
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
viability
Key result
Critical effects observed:
no
Reproductive effects observed:
not specified

Table 1: main differences in mean body weight and body weight gain (in grams)

Sex

Male

Female

Dose-level (mg/kg/day)

0

20

40

80

0

20

40

80

Pre-mating

 

 

 

 

 

 

 

 

 . days 1-71

+315

+305
(-3%)

+304
(-3%)

+315
(0%)

+133

+130
(-2%)

+125
(-6%)

+129
(-3%)

Gestation

 

 

 

 

 

 

 

 

 . days 0-20p.c.

/

/

/

/

+120

+128
(+7%)

+127
(+6%)

+112
(-7%)

Lactation

 

 

 

 

 

 

 

 

 . days 1-5 p.p.

/

/

/

/

+11

+20

(+82%)

+18

(+64%)

+13

(+18%)

/: not recorded

Table 2: delivery data

Dose-level (mg/kg/day)

0

20

40

80

Duration of gestation (days)

21.6

21.4

21.5

22.8**

. Number of females delivering on day 21p.c.

4

7

6

 

. Number of females delivering on day 22p.c.

7

4

5

2

. Number of females delivering on day 23p.c.

 

 

 

1

. Number of females delivering on day 24p.c.

 

 

 

1

Number of females surviving delivery/number of pregnant females

11/11

11/11

11/12

4/8a

Mean number ofcorpora lutea

17.0

15.7

14.9

13.2

Mean number of implantations

16.5

15.4

14.4

10.3#

Mean number of live pups delivered

14.7

14.7

13.4

9.0**

**: p<0.01, #: p<0.001.

a: excluding female K20431 which had total resorptions.

Table 3: differences (expressed in %) noted between treated and control animals in the absolute and relative organ weights

 

Sex

Male

Female

Group

2

3

4

2

3

4

Dose-level (mg/kg/day)

20

40

80

20

40

80

Body weight

-1

-2

0

-1

0

-4

- Kidneys

 

 

 

 

 

 

  . absolute

-3

-1

+13**

0

+1

-5

  . relative

-2

0

+13**

+1

+1

-1

- Liver

 

 

 

 

 

 

  . absolute

-1

+1

+15*

+1

+9

+1

  . relative

0

+3

+15**

+2

+9

+5

- Prostate

 

 

 

 

 

 

  . absolute

+4

+1

-10

 

 

 

  . relative

+4

+3

-11

 

 

 

- Seminal vesicles

 

 

 

 

 

 

  . absolute

-17*

-19*

-35**

 

 

 

  . relative

-16

-18

-35**

 

 

 

- Uterus

 

 

 

 

 

 

  . absolute

 

 

 

-3

+1

+161

  . relative

 

 

 

-1

+2

+170

Statistically significant from controls: *: p<0.05, **: p<0.01.

The significance concerned the organ weights values and not the percentages.

Table 4

Females prematurely killed or found dead

 

Dose-level (mg/kg/day)

Day*

Status**

40

80

Female

Number/status
at necropsy

Macroscopic finding

Microscopic findings

Macroscopic finding

Microscopic findings

K20415

97

Dead during pregnancy (22)

 

Dead fetuses (16)

 

Gestational uterus

Marked vaginal mucification

-

-

 

 

 

 

 

 

 

K20421

102

Dead during pregnancy (23)

-

-

Implantation scars (18)

Black thick vaginal content

Moderate vaginal mucification

 

 

 

 

 

 

 

K20422

96

Dead during pregnancy (23)

-

-

Dead fetuses (11)

Implantation scars (6)

Gestational uterus

Remnants of membranes and ombilical cord

 

 

 

 

 

 

 

K20423

98

Dead

during lactation (2)

-

-

No macroscopic observation

 

Trophoblast remnants

 

 

 

 

 

 

 

K20426

97

Dead during pregnancy (23)

-

-

Implantation scars (16)

Gestational uterus

 

 

 

 

 

 

 

K20427

98

Prematurely sacrificed during pregnancy (23)

-

-

Alive fetus (1)

Dead fetuses (15)

Late resorption (1)

Gestational uterus

 

 

 

 

 

 

 

K20429

101

Prematurely sacrificed during lactation (1)

 

 

No macroscopic observation

Slight neutrophil infiltration with necrosis and giant cells with hypertrophic nuclei

*: day of autopsy, **: gestational day or dead during lactation.

(n): number of alive or dead fetuses, late resorptions or implantation scars in uterine horns.

 


Females killed after delivery

 

Dose-level (mg/kg/day)

Day*

Status**

40

80

Female Number/status at necropsy

Macroscopic finding

Microscopic findings

Macroscopic finding

Microscopic findings

K20431

99

Final sacrifice on lactation day 5

-

-

Late resorption (1)

Thick black horn and vagina content

Minimal endometrium atrophy (epithelium and stroma)

moderate mucification of vagina

*: day of autopsy,  **: gestational day or dead during lactation.

(n): number of resorptions.

Conclusions:
Under the experimental conditions of this study:
. the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 20 mg/kg/day (based on deaths at 40 and 80 mg/kg/day),
.   male reproductive performance was not affected by treatment with sodium thioglycolate. Dosing at 40 and 80 mg/kg/day resulted in deaths in late gestation associated with delayed delivery and a No Observed Effect Level (NOEL) for female reproductive performance was therefore set at 20 mg/kg/day,
.   the NOEL for toxic effects on progeny was set at 40 mg/kg/day, based on the dead litter at 80 mg/kg/day.
A proper evaluation of the reproductive performance of females given 80 mg/kg/day was not possible because of the numerous deaths observed in this group in the peri-natal period.
Executive summary:

In a reproduction/developmental screening test performed according to the OECD Guideline # 421, four groups of 12 male and 12 female Sprague-Dawley rats received sodium thioglycolate (purity 98.9% pure), daily, by oral (gavage) administration, 10 weeks before mating and through mating and, for the females, through gestation until day 5 post-partum,at dose-levels of 0, 20, 40 or 80 mg/kg bw/d.

Clinical signs and mortality were checked daily. Body weight and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. The animals were paired for mating and the dams were allowed to litter and rear their progeny until day 5 post-partum. The total litter sizes and numbers of pups of each sex were recorded after birth, pup clinical signs were recorded daily and pup body weights were recorded on days 1 and 5 post-partum. The males were sacrificed after completion of the mating period and the females on day 5 post-partum (or on day 25 post-coitum for females which did not deliver). The body weight and selected organs (brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles and testes and uterus) were weighed and a macroscopicpost-mortemexamination of the principal thoracic and abdominal organs and a microscopic examination of selected organs (macroscopic lesions, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles, testes, and uterus) were performed. In the females, which were apparently non-pregnant, the presence of implantation scars on the uterus was checked using ammonium sulphide staining technique. Epididymal sperm was sampled for motility, morphology and count and testicular sperm heads resistant to homogenization (i.e. elongated spermatids and mature spermatozoa) were counted. The pups were sacrificed on day 5 post-partum and were carefully examined for gross external abnormalities and a macroscopicpost-mortemexamination was performed.

 

Two males (weeks 11 and 13) and one female (week 4) given 80 mg/kg/day were found dead during the pre-mating or mating periods with no clinical signs observed before death and no relevant post-mortem findings. These deaths are considered to be treatment-related. Three out of 11 surviving females given 80 mg/kg/day were found dead on day 23 post-coitum, all having delivered pups, although one female had one fetus in the vagina and still had 11 dead fetuses in the uterine horns at necropsy. Another pregnant female with dead and live fetuses in the uterine horns was sacrificed on day 23 post-coitum because of poor clinical condition. At 80 mg/kg/day, one additional female was prematurely sacrificed on day 1post-partumbecause all the pups were dead and another female was found dead on day 2 post-partum.

One female given 40 mg/kg/day was found dead on day 22 post-coitum, pregnant with dead fetuses in the uterine horns.

Ptyalism was observed at 40 and 80 mg/kg/day with a dose-related incidence and may be related to the taste of the test item.

 

There were no significant effects of treatment on mean body weight gains; all sodium thioglycolate-treated male and female groups had body weight gains similar to those of the control group throughout the study. There were no effects of treatment on mean food consumption, except a slight lowering of food consumption during the lactation period for the two females given 80 mg/kg/day (-10%).

There were no effects of treatment with sodium thioglycolate on vaginal cyclicity, with mean cycle lengths of 4 to 5 days in all groups including control. All pairs mated and the majority of the females were pregnant. There were no effects of treatment on the mean number of days taken to mate.

Females given 80 mg/kg/day had a significantly longer gestation period (22.8, p<0.01,vs. 21.6 days), a non‑significantly lower number of corpora lutea (mean of 6.7, ns, vs. 8.5) and a significantly lower number of implantations (10.3, p<0.001,vs.16.5) and pups (9.0, p<0.01, vs. 14.7). One female had total resorptions and one litter died on day 1 post-partum.

There were no treatment-related pup clinical signs or necropsy findings. Pups treated at 40 or 80 mg/kg/day had higher mean body weight gains than the controls between day 1 and day 5 post‑partum.

There were no effects of treatment on sperm morphology, motility or counts. The mean liver and kidneys weights were slightly but statistically significantly higher for males given 80 mg/kg/day (+15% for liver and +13% for kidneys in absolute weights). Higher liver weights correlated with a trend towards increased glycogen content at this dose-level and was considered to be related to the test item administration.For the higher kidney weights, a relationship to treatment was considered to be equivocal as there were no histopathological correlates.The mean absolute seminal vesicle weights were statistically significantly lower for all male groups in a dose-related manner (absolute weights were -17%, -19% and -35% at 20, 40 and 80 mg/kg/day, respectively). This correlated with a slight decrease in secretory content in the seminal vesicles observed at microscopic examination of the males given 80 mg/kg/day. In the absence of atrophy of seminal epithelium at microscopic examination, these minor findings were not considered to be adverse.

 

The dose-level of 80 mg/kg/day was considered to be higher than the Maximum Tolerated Dose for a dosing period of 13 weeks as there were two males and one female found dead during the premating or mating periods. In addition, treatment at this lethal dose was associated with delayed delivery as four females were found dead or prematurely sacrificed after the normal period for delivery and had not delivered all the pups. Administration of sodium thioglycolate at this lethal dose of 80 mg/kg/day was also associated with the death or premature sacrifice of two additional females during the peri-natal period (from day 1 to day 2post-partum). The female sacrificed was killed on day 1post-partumbecause all its litter died. There were no effects on male or female mating behavior or fertility and the test item was considered not to hinder embryo-fetal development. The test item at the mid and high dose-levels caused increased pup body weight gain after birth but there were no relevant pup clinical signs orpost-mortemfindings. In males, when compared to controls, liver and kidneys weights were slightly but significantly higher. There were no relevant macroscopic or microscopic findings nor any effects on sperm morphology, motility or counts.

At 40 mg/kg/day, one pregnant female with dead fetuses in the uterine horns was found dead on day 22post-coitum. There were no effects of treatment on body weight, food consumption, male or female mating behavior and fertility and pregnancy parameters. There were no adverse effects of treatment on the pup body weight gain after birth.

There were no effects of treatment at 20 mg/kg/day.

Under the experimental conditions of this study:

.   the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 20 mg/kg/day (based on deaths at 40 and 80 mg/kg/day),

.   male reproductive performance was not affected by treatment with sodium thioglycolate. Dosing at 40 and 80 mg/kg/day resulted in deaths in late gestation associated with delayed delivery and a No Observed Effect Level (NOEL) for female reproductive performance was therefore set at 20 mg/kg/day,

.   the NOEL for toxic effects on progeny was set at 40 mg/kg/day, based on the dead litter at 80 mg/kg/day.

A proper evaluation of the reproductive performance of females given 80 mg/kg/day was not possible because of the numerous deaths observed in this group in the peri-natal period.

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 February 2009 - 13 October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
the temperature and the relative humidity were sometimes outside the target range + deviations to study plan
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
yes
Remarks:
idem above
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 440/2008 B.35
Deviations:
yes
Remarks:
idem above
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: the males were approximately 6 weeks old and the females were approximately 5 weeks old
- Weight: males: mean body weight of 175 g (range: 156 g to 202 g) /females: mean body weight of 109 g (range: 92 g to 125 g)
- Fasting period before study: no
- Housing: the F0 males and females and the F1 generation after weaning were individually housed, except during pairing, in wire-mesh cages.
A metal tray containing autoclaved sawdust was placed under each cage.
Towards the end of the gestation period, and with their litter during lactation, the F0 and F1 females were housed in polycarbonate cages containing
autoclaved sawdust. Autoclaved wood shavings were provided as nesting material, a few days before delivery and during the lactation period.
- Diet (e.g. ad libitum): all animals had free access to SSNIFF R/M-H pelleted maintenance diet distributed weekly
- Water (e.g. ad libitum): the animals had free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): 12 h cycles/hour
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

IN-LIFE DATES: From: 10 February 2009 To: 21 October 2009.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified
Details on exposure:
VEHICLE
- Nature: degassed purified water obtained by reverse osmosis and subsequently degassed by sonication for at least 15 minutes and finally saturated
with nitrogen gas for at least 15 minutes. This was stored under nitrogen atmosphere.
- Concentration in vehicle: 2, 4 and 8 mg a.i./mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution. The required quantity of test item was mixed with the required quantity of vehicle in order to prepare a
solution at the highest required concentration (8 mg a.i./mL). The low and intermediate concentrations (2 and 4 mg a.i./mL) were prepared by dilution of the high concentration with the vehicle.
The test item dosage forms were prepared weekly by the CIT Pharmacy under nitrogen atmosphere and were stored, in brown glass bottles, at +4°C
and under nitrogen atmosphere until treatment.
All concentrations and dose-levels in this study are expressed as active ingredient.

Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: until mating occurred or 14 days had elapsed
- Proof of pregnancy: vaginal plug or sperm in vaginal lavage (day of confirmed mating was designated day 0 post-coitum)
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): housed individually in polycarbonate cages.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Method: HPLC/UV
The test item concentration in samples of each control and test item dosage form prepared for use in weeks 1, 4, 8, 12, 16, 20, 24, 28, 32 and 36 was determined.
Duration of treatment / exposure:
Each animal was given the appropriate dosage form once a day, at approximately the same time each day, 7 days a week, according to the following
schedule:
- in the males:
- 10 weeks before mating,
- during the mating period (up to 3 weeks),
- until sacrifice (after weaning of the pups).

- in the females:
- 10 weeks before mating,
- during the mating period (up to 3 weeks),
- during pregnancy,
- during lactation until day 21 post-partum inclusive,
- females with no delivery were treated until the day prior to sacrifice.

Day 1 corresponds to the first day of treatment period.
Frequency of treatment:
Once daily.
Details on study schedule:
- F1 parental animals not mated until 9 to 11 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 22 days of age.
- Age at mating of the mated animals in the study: between 12 and 14 weeks
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
For dose-levels of 0, 10 and 20 mg a.i./kg/day: 25 animals per sex and per dose
For dose-level of 40 mg a.i./kg/day: 25 (P0) or 27 (F1) animals per sex.
Control animals:
yes
Details on study design:
- Dose selection rationale:
The dose-levels were selected on the basis of the results of previous studies:
- an OECD 421 study using dose-levels of 20, 40 and 80 mg/kg/day (CIT/Study No. 30721 RSR) in which the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 20 mg/kg/day (based on deaths at 40 and 80 mg/kg/day). Male reproductive performance was not affected by treatment with sodium thioglycolate. Dosing at 40 and 80 mg/kg/day resulted in deaths in late gestation associated with delayed
delivery and a No Observed Effect Level (NOEL) for female reproductive performance was therefore set at 20 mg/kg/day. The NOEL for toxic effects on progeny was set at 40 mg/kg/day, based on the dead litter at 80 mg/kg/day.
- a 13-week toxicity study in rats using dose-levels of 7, 20 and 60 mg/kg/day (CIT/Study No. 38414 TCR) in which mortality occurred at
60 mg/kg/day and a few hematology, blood biochemistry and microscopic effects were observed at 20 mg/kg/day.

- Rationale for animal assignment: random.
Positive control:
None.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: the body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice.
The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and
on days 0, 7, 14 and 20 post-coitum and days 1, 4, 7, 14 and 21 post-partum.
The female prematurely sacrificed was weighed prior to sacrifice.

FOOD CONSUMPTION: once a week
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No.

LABORATORY INVESTIGATIONS (F0 generation)
- Blood sampling
Blood samples were taken from the orbital sinus of non-fasted animals (5 to 6 hours after treatment) under light isoflurane anesthesia, and collected into tubes containing the appropriate anticoagulant. All samples for hematology, blood biochemistry and β-hydroxybutyrate and acetoacetate analysis were taken at the same moment for the males. The females were sampled either on day 21 post partum or on day 24 post-coitum if no delivery had occurred
- Hematology: peripheral blood
The following parameters were determined for all surviving animals towards the end of the treatment period, 5 to 6 hours after treatment, on day 24 post-coitum for all females not having delivered and for moribund animal T20401 (4F) prior to sacrifice on day 1 post-partum.
Erythrocytes (RBC)
Hemoglobin (HB)
Mean cell volume (MCV)
Packed cell volume (PCV)
Mean cell hemoglobinconcentration (MCHC)
Mean cell hemoglobin (MCH)
Thrombocytes (PLT)
Leucocytes (WBC)
Differential white cellcount with cell morphology
. neutrophils (N)
. eosinophils (E)
. basophils (B)
. lymphocytes and large unstained cells (L+LUC)
. monocytes (M)
Prothrombin time (PT)
- Blood biochemistry
The following parameters were determined for all surviving animals towards the end of the treatment period, 5 to 6 hours after treatment, on day 24 post-coitum for all females not having delivered and for moribund animal T20401 (4F) prior to sacrifice on day 1 post-partum.
Chloride (Cl-)
Glucose (GLUC)
Urea (UREA)
Creatinine (CREAT)
Triglycerides (TRIG)
Aspartateaminotransferase (ASAT)
Alanineaminotransferase (ALAT)
Free fatty acids (ACGR)
Lactate (LACT)
β hydroxybutyrate
acetoacetate
Estrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning as follows:
- during the last 3 weeks of the pre-mating period,
- during the mating period, until the females were mated.
Sperm parameters (parental animals):
Parameters examined in F0 and F1 male parental generations (on the first ten surviving F0 males and the first ten surviving F1 males of the control
and high-dose groups):
testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal
sperm reserve, sperm motility, sperm morphology.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 post-partum: yes
- If yes, maximum of 8 male and 8 female pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring of F0 and F1 generations: number and sex of pups, stillbirths, live births, postnatal mortality,
presence of gross anomalies, weight gain, physical or behavioural abnormalities, other: reflex development, physical development, presence of
nipples in males (progeny of F1).

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities in:
- pups found dead,
- pups prematurely sacrificed,
- pups culled on post natal day 4 (PND 4),
- pups sacrificed on PND 22.

yes, for internal abnormalities in:
- pups showing external abnormalities or clinical signs,
- pups found dead or prematurely sacrificed,
- one randomly selected F1 and F2 pup/sex/litter sacrificed on PND 22.

Possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: in all surviving animals, after weaning of the litters
- Maternal animals: in all surviving animals, at the weaning of the litters. Females which did not deliver were sacrificed on day 25 post-coitum after body weight recording. Females with litter dying entirely were sacrificed as appropriate

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 (table procedure) were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed on PND 4 and PND 22, all F2 offspring were sacrificed at the weaning of the litters.
- The following animals were subjected to postmortem examinations (gross external abnormalities):
. pups found dead,
. pups prematurely sacrificed,
. pups culled on PND 4,
. pups sacrificed on PND 22.

GROSS NECROPSY (progeny of the F0 and F1 generations)
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Gross necrospy was performed in:
. pups showing external abnormalities or clinical signs,
. pups found dead or prematurely sacrificed,
. one randomly selected F1 and F2 pup/sex/litter sacrificed on PND 22.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 2 were prepared for microscopic examination and weighed, respectively.
Reproductive indices:
Post-implantation loss:
Number of implantation sites - Number of live pups
_____________________________________________ x 100
Number of implantations

Mating index:
Number of mated animals
_____________________ x 100
Number of paired animals

Fertility index:
Number of pregnant females
_______________________________ x 100
Number of mated females

Gestation index:
Number of females with live born pups
________________________________ x 100
Number of pregnant females
Offspring viability indices:
Live birth index:
Number of live born pups
_____________________ x 100
Number of delivered pups

Viability index on day 4 post-partum:
Number of surviving pups on day 4 post-partum
_______________________________________ x 100
Number of live born pups

Lactation index on day 21 post-partum:
Number of surviving pups on day 21 post-partum
________________________________________ x 100
Number of surviving pups on day 4 post-partum
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see below
Mortality:
mortality observed, treatment-related
Description (incidence):
see below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see below
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: estrous cycle:
effects observed, treatment-related
Description (incidence and severity):
see below
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
see below
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no premature deaths in the groups treated at 0, 10 or 20 mg a.i./kg/day.
At 40 mg a.i./kg/day, four females were found dead; one on gestation day 21 (T20408) and three on gestation day 22 (T20410, T20418 and
T20420). None of the females had clinical signs prior to death except that female T20420 had just completed delivery and had given birth to
12 live pups and one dead pup. At necropsy there were 13 implantation scars on the uterine horns which matches the number of pups delivered. The other females had not started delivery and had dead fetuses in the uterine horns at necropsy.
Female T20420, who had just delivered, had a hemorrhage of one mesometrial triangle in the uterus which could have contributed to the death.
In females T20410 and T20418 some mesometrial triangles were present in the histological sections of the uterus but there were no microscopic
findings including hemorrhage which could explain the deaths.
Another female treated at 40 mg a.i./kg/day (female T20401) was prematurely sacrificed on lactation day 2 because all pups were cannibalized on
lactation day 1. The female still had piloerection, blood, placentae and fetuses in the bedding (but not in the uterine horns) on lactation day 2
indicating poor clinical condition of the dam after the pups had been born. At microscopic examination, septicemia, peritonitis and abscesses in
the mesometrial triangles, thought to be of uterine origin, were observed.
It is concluded that the test item causes mortality of susceptible dams around the time of delivery. In a few females which deliver, nesting/nursing
behavior is impaired which causes the pups to die or be cannibalized.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In males, it was considered that there were no effects of treatment with the test item.
All female test item-treated groups had similar mean body weight gains to the controls during the pre-pairing, gestation and lactation phases,
except during the first 4 days of lactation where the group treated at 40 mg a.i./kg/day had a statistically significantly lower mean body weight gain
(5 g vs. 13 g, p<0.01). There were three females in group 4 which lost weight during this period but the majority of the females in this group gained
little weight. This may be related to difficulties at time of delivery and a longer recovery time at this dose-level.
The mean food consumption of males and females treated with test item was comparable with that of the controls throughout the premating,
gestation and lactation periods at all dose-levels.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The mean number of cycles per female was minimally lower at all dose-levels when compared with the controls. This is partly due to the females
with abnormal cycling, although there were not sufficient numbers of females per group nor a dose-relationship to conclude that there was a
treatment-related effect.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no effects of treatment with the test item on sperm parameters.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No problems were encountered during the mating period; all groups mated within a mean of 3 days and the number of pregnant females in each group was considered to be within normal limits.
At 40 mg a.i./kg/day, four females were found dead on gestation day 21 or 22 and another female (T20412) did not deliver and was found to have only implantation scars at necropsy. The body weight of this female 20 days after mating was similar to that of non-pregnant females so it was considered that the implantation scars remained after early resorptions had occurred.
The mean number of implantation sites was similar among all groups. The post-implantation loss and the mean number of pups delivered were minimally higher or lower, respectively, in all test item-treated groups when compared with the controls (the high-dose group was outside historical data range for both the post-implantation loss and the number of pups born but in the absence of a significant difference when compared with the controls of this study, this was considered not to be relevant). Female T20401 (40 mg a.i./kg/day) had five more implantation sites than pups born but was prematurely sacrificed on lactation day 2 because of cannibalism of the litter on lactation day 1. The implantation sites were counted at necropsy but there were no resorptions. It is likely that this female delivered five pups which were never seen because they were cannibalized after birth. In this case, the post-implantation loss is skewed by differences in the pup numbers that were not due to mortality in utero. At the dose-levels of 10 and 20 mg a.i./kg/day respectively, female T20366 had 12 fewer pups than implantation sites and female T20395 had six fewer pups than implantation sites. Overall, it was considered that there were no effects of treatment on in utero implantation loss.
A high number of pups died in the group treated at 40 mg a.i./kg/day. Of the 21 pups which died during lactation, 16 were from two litters. Female T20401, who had piloerection and hypoactivity before delivery and piloerection and blood, fetuses and placentas in the bedding for several days after delivery, cannibalized all the pups on lactation day 1. It is not possible to know whether the pups were born healthy and correctly formed or whether they died before being cannibalized since a macroscopic examination was not possible. Another six of the dead pups were from female T20404 who showed no clinical signs but cannibalized two pups (the other four were found dead, one having shown pallor on the day prior to death). The remaining five pups were scattered among five different litters.
The five dead pups at 20 mg a.i./kg/day were from five different litters as were the 13 dead pups at 10 mg a.i./kg/day.
Overall, the pup deaths in the control group and the group treated at 20 mg a.i./kg/day were scattered one pup per affected litter, but at 10 and 40 mg a.i./kg/day the deaths tended to be concentrated in two litters. With the exception of female T20401 discussed above, there was nothing remarkable about these females in terms of clinical signs, gestational body weight or number of pups. Some cannibalized pups were cold to the touch and pale the day before death but the found dead pups, with one exception, did not show any clinical signs prior to death.

LABORATORY INVESTIGATIONS
- Hematology
Males treated at 40 mg a.i./kg/day had a statistically significantly increased hemoglobin level in the blood when compared with the controls (15.5 g/dL vs. 15.0 g/dL, p<0.05). This treatment related increase was not observed in the females treated at the same dose-level. None of the other red blood cell parameters showed differences to the controls so a relationship to treatment of this isolated parameter is doubtful and of no biological significance. In addition, the hemoglobin concentration was within Historical data range for male rats of this approximate age (13.3 to 17.2 g/dL).
- Blood biochemistry

Sex Male Female
Dose-level (mg a.i/kg/day) 0 10 20 40 0 10 20 40
Urea(mmol/L) 5.2 4.9 4.8 4.7* 8.4 8.4 7.9 8.2
Fatty acids(mmol/L) 0.08 0.10 0.10 0.08 0.07 0.06 0.05 0.04**
Acetoacetate(μmol/L) 57.3 55.4 57.1 63.7 58.2 55.7 61.0 65.4
ß-hydroxybutyrate(μmol/L) 64.3 64.8 59.8 56.8 62.7 61.1 62.0 63.5
Statistically significant *: p<0.05, **: p<0.01.

Males treated at 40 mg a.i./kg/day had a statistically significantly decreased urea concentration when compared with the controls, but of no biological significance. Females treated at the same dose-level had a statistically significantly decreased fatty acid concentration when compared with the controls. Neither of these differences was observed in the other sex or at the other dose-levels.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No changes in organ weights attributable to the treatment with Sodium thioglycolate were observed in parents.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Unscheduled death
Females T20408 and T20410, given the test item at 40 mg a.i./kg/day were found dead on days 21 and 22 of gestation, respectively. No abnormalities were observed at necropsy but 15 and 14 dead fetuses in the uterus, respectively. The cause of the death could not be determined.
Female T20418 given 40 mg a.i./kg/day was found dead on day 22 of gestation. At necropsy, 15 dead fetuses were observed in the uterus. Pinpoint
black discolored foci were seen in the stomach without microscopic correlates.
Female T20420 given 40 mg a.i./kg/day was found dead on day 22 of gestation. The uterus presented 13 implantation scars correlating to the
13 pups found in the cage. No macroscopic findings could explain the death of the rat.
Female T20401, given the test item at 40 mg a.i./kg/day was prematurely sacrificed on day 2 of lactation because all the pups had been cannibalized
on day 1 of lactation. At necropsy, the kidneys were green and correlated microscopically with capsular acute inflammation. The liver showed an
irregular color and the caudate lobe presented a pouch that contained a thick green content. It correlated microscopically with a marked acute
capsular inflammation infiltrating the hepatic parenchyma. These macroscopic findings correlated with the septicemia and peritonitis noted at
microscopy. The adrenals were enlarged with white discoloration that correlated at microscopic examination with cortical hyperplasia and cortical
necrosis, respectively. Thymus was small. These changes were attributed to the stress associated with the poor clinical condition.
The liver had an accentuated lobular pattern that correlated microscopically with the periportal hepatocellular microvacuolation (cf terminal sacrifice, liver microscopic examination) and with the slight hepatocellular glycogen content.

Terminal sacrifice
No treatment-related macroscopic findings were observed in F0 parents given Sodium thioglycolate at 10, 20 or 40 mg a.i./kg/day.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Unscheduled death
Treatment-related changes were seen in the uterus from some prematurely sacrificed/found dead females treated at 40 mg a.i./kg/day and could be related to mortality around the time of delivery.
Females T20408, T20410 and T20418, given the test item at 40 mg a.i./kg/day were found dead on day 21, 22 and 22 of gestation, respectively. No
microscopic findings could explain the death of these animals. Female T20410 and T20418 presented in the uterus some mesometrial triangles. This normal structure of both fetal/maternal origin is part of the placenta that infiltrates the uterine wall (rat hemochorial placentation) and is involved in the normal gestation. It is characterized by invasion of trophoblastic cells in the uterine wall (mesometrium, myometrium and endometrium), forming large "lakes" of blood, consisting in vascular structures lined by inconstant bumpy cells and glycogen type cells. The interstitium in these locations presented glycogen type, trophoblastic and granulated cells.
Female T20420 given 40 mg a.i./kg/day was found dead on day 22 of gestation. At microscopic examination, mesometrial triangles were seen in the uterus along with minimal hyperplasia of the endometrial epithelium. In the center of one of these mesometrial triangles, slight necrosis of the endometrium (stroma, epithelium and glands) was observed with subsequent hemorrhage that collected in the lumen of the uterus and that was also found in the lumen of the vagina. This bleeding could have contributed to the death of the rat. A relationship to treatment could not be ruled out.
Female T20401, treated at 40 mg a.i./kg/day and prematurely sacrificed on day 2 of lactation presented microscopic findings suggestive of septicemia and peritonitis. The surface of the liver and, at a lesser severity of the kidneys, presented a diffuse acute suppurative and necrotic inflammation with presence of bacterial colonies. These findings correlated with the irregular discoloration and green discoloration of these two organs seen at necropsy along with the green pouch seen in the liver. In the liver, the inflammation extended into the adjacent parenchyma, resulting in hepatocellular necrosis and infiltration of the parenchyma by mixed inflammatory cells. Minimal multifocal cortical necrosis of the left adrenal correlated with the white discoloration seen macroscopically. The origin of the inflammatory process was though to be the uterus. In the uterus, severe abscessation of the mesometrial triangle was seen in continuation with the outer layer of the uterus. It consisted of very large abscesses characterized by central coagulation necrosis of the vascular and connective tissues, degenerated neutrophils and fibrin exudation. Bacterial colonies were numerous within the abscesses and in vascular structure (bacteremia / septicemia). Mesothelial cells are bumpy and prominent (hyperplasia and hypertrophy). No luminal inflammation (pyometra) was evident but necrotic debris of the endometrial epithelium along with degenerated
neutrophils and fibrin can be seen. This inflammatory change was also associated with mesometrial thrombosis in the abscesses and in the adjacent tissue and with slight necrosis of the superficial and glandular endometrial epithelium. A hemorrhagic collection from these lesions was seen in the lumen of uterus and drained through the cervix into the vagina. Adrenal cortical hyperplasia and severe lymphoid atrophy in the thymus were attributed to the stress associated with the poor clinical condition. These findings had contributed to the poor clinical condition of the female. In the absence of control animals at a similar stage of gestation, a relationship between the uterine lesions and the treatment with Sodium thioglycolate could not be ruled out, although the infection seen in the uterus could be a contributing factor.

Terminal sacrifice
Males F0
No treatment-related microscopic changes were noted in the testis, epididymis, prostate, coagulating glands, or seminal vesicles in males given
40 mg a.i./kg/day.

Females F0
Changes that could be related to treatment were seen in the uterus of one female given 40 mg a.i./kg/day.
Qualitative evaluation of the ovaries, uterus and vagina
The histomorphological characteristics of the estrous cycle were present in almost the equal of treated and control females (11/20 versus 15/25,
respectively). Microscopic findings suggestive of delayed / disturbance of estrous cycle (mucification of the vaginal epithelium), suggesting that they had not started cycling again, were present in 10/25 control females and 9/20 treated females.
Minimal to slight greenish pigment laden macrophages located in the endometrium, myometrium and mesometrium were seen in 18/25 control
females and in 10/20 treated females. These macrophages were frequently associated with minimal to moderate remnant of the mesometrial triangle. These mesometrial remnants were seen in 21/25 control females and 14/20 treated females.
In one female (T20412) given 40 mg a.i./kg/day, thrombosis of the mesometrial remnant organized by a well developed granulation tissue composed of fibroplasia, fibrin plugs, thrombi and neovascularization (a large part of these vessels most likely belonged to the placentation). Golden pigment
laden macrophages were numerous. Pyknotic trophoblastic cells were seen. This granulation tissue probably corresponded to a scar of the
placentation that correlated with implantation scars seen at necropsy. These changes were suggestive of early resorption. As no control female was
sacrificed at the same period of the study, a definitive relationship could not be stated.

From the quantitative analysis (ovaries), minimal changes of the number of primordial follicles and corpora lutea were observed in the treated females. The minor differences were due, at least in part, to the inter-individual variability.

Treatment-related microscopic change was seen in the liver of both males and females given 40 mg a.i./kg/day and consisted of periportal hepatocellular microvacuolation.
Minimal to moderate periportal hepatocellular microvacuolation was noted in the liver of 2/25 males and 6/25 females treated at 40 mg a.i./kg/day.
Among affected females, 4/6 were found dead or prematurely sacrificed and 2/6 were sacrificed at the end of the treatment period. This finding was
characterized by densely packed microvacuoles within the cytoplasm and correlated with the accentuated lobular pattern noted in one female at
necropsy. This change most likely corresponded to steatosis (neutral lipids). Considering the very low incidence in males given 40 mg a.i./kg/day, a
relationship to treatment was uncertain. In females, the incidence and severity were higher than in males and although the affected females were
mainly the prematurely sacrificed or found dead females, a relationship to treatment could not be excluded.
Minimal increased incidence of hepatocellular degeneration/necrosis was seen in treated females. Due to the multifocal isolated distribution of limited areas, a relationship to treatment was considered unlikely.
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Key result
Dose descriptor:
LOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Key result
Critical effects observed:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see below
Mortality:
mortality observed, treatment-related
Description (incidence):
see below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see below
Food efficiency:
not specified
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: estrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
see below
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One control male (T20115) was found dead on day 46 of treatment without having shown any clinical signs prior to death. This male had no macroscopic or microscopic findings that could have contributed to the death but did have marked infiltration of the prostatic interstitium by mononuclear inflammatory cells.
Two females treated at 40 mg a.i./kg/day (T20518 and T20520) were sacrificed on days 2 or 5 of lactation, respectively, due to dead litter. Neither dam showed clinical signs although the pups of female T20520 were cold to the touch indicating possibly poor maternal nesting behavior.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
F1 generation:
There were no effects of treatment with the test item on mean male body weight or body weight gain the during the 10-week premating treatment period.
Females treated at 40 mg a.i./kg/day started the F1 generation with a statistically significantly lower mean body weight than the controls. The female pups had finished lactation with a mean body weight which was 4% lower than that of the controls. After selection the mean body weight was 7% lower than that of the controls. Mean body weight gain during the first week of treatment was statistically significantly lower than that of the controls and the lower mean body weight continued until the fourth week of treatment, by which time it was only slightly lower than that of the controls and it remained minimally lower until the end of the premating period (-3% on day 71). During the gestation period, females treated at 40 mg a.i./kg/day gained less weight than the controls and had a mean body weight difference of -5% on gestation day 20, however greater body weight gains during lactation reduced the deficit.
It is considered that there was a minimal effect of treatment with the test item on body weight of the F1 generation females at 40 mg a.i./kg/day.
There were no effects on mean male food consumption.
Females treated at 40 mg a.i./kg/day had a statistically significantly lower mean food consumption during the first week of treatment (13 g/animal/day, p<0.05, vs. 15 g/animal/day), but thereafter mean food intake was comparable with the controls.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no effects of treatment with the test item on estrous cycles.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no effects of treatment with the test item on sperm parameters.
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
One male treated at 10 mg a.i./kg/day and three males treated at 40 mg a.i./kg/day did not mate during the 14 days of pairing with the female. All females were re-paired with males that had previously mated and mating occurred within 5 days. The number of males not mating at 40 mg a.i./kg/day was rather high (male mating index = 89%, compared to the background data mean of 99%). Males T30139 (10 mg a.i./kg/day), T20194 and T20197 (40 mg a.i./kg/day) had no abnormal macroscopic findings at necropsy but male T20183 (40 mg a.i./kg/day) had small right testis and epididymis at necropsy and little seminal liquid with virtually no spermatozoa at sperm analysis.
Five females treated at 40 mg a.i./kg/day mated but were not pregnant. Two of the females were not cycling (in diestrus for 9 or 12 days) and one female was paired with a male which was later found to have aspermia/oligospermia. None of the other males had microscopic findings which were considered to have impaired fertility.
Overall, the time taken to mate was prolonged in these two groups because of the males which did not mate but it was considered that treatment with the test item did not specifically delay mating.
The mean duration of gestation was comparable to that of the controls for all groups.
The mean numbers of implantation sites and pups born were comparable with the controls at all dose-levels. The mean post-implantation loss at 10 and 20 mg a.i./kg/day was slightly higher than the controls, and outside Historical data range, but the group treated at 40 mg a.i./kg/day had a lower mean post-implantation loss than the controls, and was within Historical data range, so it was considered that this increase at the low and intermediate dose-levels was incidental.
At 40 mg a.i./kg/day, two entire litters died. Female T20518 had only one pup which died on lactation day 2 without any observed clinical signs. There were no recorded implants in the uterine horns at sacrifice although there were greenish pigmen-laden macrophages and necrosis of the endometrium in the uterus.
Female T20520 had 16 pups; one was found dead on lactation day 2, two were cannibalized on lactation day 3, four were cannibalized and two were found dead on lactation day 4 and the remaining seven were cannibalized on lactation day 5. The majority of the pups were observed to be cold on lactation day 3 which indicated lack of nesting/nursing by the dam or inability or lack of desire to nurse in the pups. The dam showed no signs of poor clinical condition.
The remaining 14 dead pups from the 40 mg a.i./kg/day group were spread between seven litters (between one and five dead pups per litter).
Four litters (between one and eight dead pups per litter). The groups treated at 10 or 20 mg a.i./kg/day had fewer dead pups than the control group.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No changes in organ weights attributable to the treatment with Sodium thioglycolate were observed in F1 parents.
GROSS PATHOLOGY (PARENTAL ANIMALS)
Unscheduled death
One control male was found dead on day 46. No macroscopic findings that could have contributed to the death of the rat were observed at necropsy.
Females T20518 and T20520 were prematurely sacrificed on days 2 and 5 of lactation, respectively, due to dead litter. No abnormalities were observed at necropsy.
Terminal sacrifice
No treatment-related macroscopic findings were observed in F1 parents given Sodium thioglycolate at 10, 20 or 40 mg a.i./kg/day.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Unscheduled death
Control male T20115, found dead on day 46, presented marked mononuclear inflammatory cells infiltration of the prostatic interstitium. No microscopic findings that could have contributed to the death of the animal were observed.
Female T20518, prematurely sacrificed on day 2 of lactation, presented moderate multifocal remnants of mesometrial triangles, indicative of gestation. The uterus of female T20520, given 40 mg a.i./kg/day and prematurely sacrificed on day 5 of lactation, showed large mesometrial triangles. The mesometrial triangles presented thrombosis and were largely replaced by granulation tissue. These changes could be related to difficulties seen during delivery that could be treatment-related.

Terminal sacrifice
Males F1
No treatment-related microscopic changes were noted in the testis, epididymis, prostate, coagulating glands, or seminal vesicles in males given 40 mg a.i./kg/day.
Females F1
No treatment-related microscopic changes were noted in ovaries, oviducts, uterus or vagina in females given 40 mg a.i./kg/day.
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Key result
Dose descriptor:
LOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Key result
Critical effects observed:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see below
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
see below
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see below
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
VIABILITY (OFFSPRING)

The pup deaths in the control group and the group treated at 20 mg a.i./kg/day were scattered one pup per affected litter, but at 10 and 40 mg a.i./kg/day the deaths tended to be concentrated in two litters.

Dose-level (mg a.i./kg/day) 0 10 20 40
Number of cannibalized pups 1 6 2 13
Number of cannibalized pups with clinical signs 1 4 0 0
Number of pups found dead 4 6 3 8
Number of found dead pups with clinical signs 0 0 0 1
Number of prematurely sacrificed pups 0 1a 0 0
a: pup 14 of female T20360 with necrosed right hindlimb, pallor, thin appearance.


CLINICAL SIGNS (OFFSPRING)
Some cannibalized pups were cold to the touch and pale the day before death but the found dead pups, with one exception, did not show any clinical signs prior to death.

BODY WEIGHT (OFFSPRING)
There were no effects of treatment with the test item on mean body weight or body weight gain in F1 and F2 generations.

SEXUAL MATURATION (OFFSPRING)
It was considered that the difference in age at balanopreputial separation was not related to treatment with the test item.
It was considered that there were no effects on the age of vaginal opening at any dose-level.

ORGAN WEIGHTS (OFFSPRING)
No changes in organ weights attributable to the treatment with Sodium thioglycolate were noted in F1 and F2 pups.

GROSS PATHOLOGY (OFFSPRING)
There were no relevant findings at necropsy in F1 pups found dead or sacrificed at weaning at any dose-level.
No treatment-related macroscopic findings were noted in F2 pups.

HISTOPATHOLOGY (OFFSPRING)
No treatment-related observations were noted in F1 and F2 pups.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
viability
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
viability
Key result
Critical effects observed:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see below
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
see below
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not specified
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:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
VIABILITY (OFFSPRING)
At 40 mg a.i./kg/day, two entire litters died. Female T20518 had only one pup which died on lactation day 2 without any observed clinical signs.
Female T20520 had 16 pups; one was found dead on lactation day 2, two were cannibalized on lactation day 3, four were cannibalized and two were found dead on lactation day 4 and the remaining seven were cannibalized on lactation day 5.
The remaining 14 dead pups from the 40 mg a.i./kg/day group were spread between seven litters (between one and five dead pups per litter). In the control group, the 16 dead pups were spread between four litters (between one and eight dead pups per litter). The groups treated at 10 or 20 mg a.i./kg/day had fewer dead pups than the control group.

Dose-level (mg a.i./kg/day) 0 10 20 40
Number of cannibalized pups 10 4 4 16
Number of cannibalized pups with clinical signs 4 1 0 14
Number of pups found dead 6 5 7 15
Number of found dead pups with clinical signs 4 3 2 4
Number of prematurely sacrificed pups 0 0 2a 0
a: female T20481: pups 3 and 10 because of tremors, locomotory difficulties, piloerection and emaciation.

CLINICAL SIGNS (OFFSPRING)
The majority of the cannibalized pups were observed to be cold on lactation day 3 which indicated lack of nesting/nursing by the dam or inability or lack of desire to nurse in the pups. In the litters where cannibalized and found dead pups occurred, the other pups often showed the same clinical signs, generally coldness.

BODY WEIGHT (OFFSPRING)
There were no effects of treatment with the test item on mean body weight or body weight gain in F1 and F2 generations.

SEXUAL MATURATION (OFFSPRING)
It was considered that the difference in age at balanopreputial separation was not related to treatment with the test item.
It was considered that there were no effects on the age of vaginal opening at any dose-level.

ORGAN WEIGHTS (OFFSPRING)
No changes in organ weights attributable to the treatment with Sodium thioglycolate were noted in F1 and F2 pups.

GROSS PATHOLOGY (OFFSPRING)
No treatment-related macroscopic findings were noted in F2 pups.

HISTOPATHOLOGY (OFFSPRING)
No treatment-related observations were noted in F1 and F2 pups.
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
viability
Key result
Dose descriptor:
LOAEL
Generation:
F2
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
viability
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
40 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Sodium thioglycolate, was administered once daily by oral gavage to males and females for 10 weeks prior to pairing, during mating, gestation and lactation until weaning of the pups. Selected pups formed the F1 generation and were treated with the test item once daily by oral gavage for 10 weeks, from day 22 of age until the start of the pairing period, during mating, gestation and lactation until weaning of the F2 pups. The dose-levels for both generations were 10, 20 and 40 mg a.i./kg/day (a.i. = active ingredient). Due to mortalities around the time of delivery in the F0 females, the F1 generation females were not treated from gestation day 19 until lactation day 1.
At 40 mg a.i./kg/day, it is concluded that sodium thioglycolate has no effect on non-pregnant, naïve, adult rats but that it causes maternal toxicity and death of susceptible pregnant females around the time of delivery. Effects on the dam include lack of nesting/nursing behavior and this causes death of the pups which have been delivered. If treatment is stopped just prior to delivery, the females may survive delivery but pup death may still occur and pup clinical signs of coldness suggest that maternal nesting/nursing behavior is still impaired by treatment with the test item and affect pup survival.
There is evidence that female rats are more affected by sodium thioglycolate treatment than males as shown by lower F1 female body weight and body weight gain.
Minimal to moderate periportal heptocellular microvacuolation was observed in F0 animals treated at 40 mg a.i./kg/day suggestive of mild
hepatotoxicity and especially in dams found dead or prematurely sacrificed at the time of parturition (out of the eight animals presenting this finding, four were found dead or prematurely sacrificed dams). Sodium thioglycolate is known to induce fatty liver via an inhibition of the β-oxidation of fatty acids.

There were no effects on sperm parameters in the control or high-dose group males of either generation.
There were no effects of treatment on any parameters measured in either males or females with the test item at 10 or 20 mg a.i./kg/day.

Under the experimental conditions of this study, and in view of the maternal mortalities and liver effects in males and females observed at 40 mg a.i./kg/day, the dose-level of 20 mg a.i./kg/day was considered to be the No Observed Effect Level (NOEL) for parental toxicity, female fertility and gestation of each generation and for development, growth and survival of the progeny. It is probable that the small effects observed on pup survival at 40 mg a.i./kg/day were secondary to the severe and lethal effects observed in the pregnant dams at that dose level.

The No Observed Effect Level (NOEL) for males fertility and female mating behaviour was higher or equal than 40 mg a.i./kg/day.
Executive summary:

The objective of this study was to provide general information concerning the effects of the test item,Sodium thioglycolate,on the integrity and performance of the male and female reproductive systems, including gonadal function, the estrous cycle, mating behavior, conception, gestation, parturition, lactation and weaning, and on the growth and development of the offspring over 2 generations. This study was performed under GLP compliance and following the OECD Guideline for Testing of Chemicals No. 416 (Two-Generation Reproduction Toxicity Study), 22ndJanuary 2001.

Three groups of 25 male and 25 female Sprague-Dawley rats received the test item, Sodium thioglycolate (batch No.25462), daily for 10 weeks prior to mating, during mating, gestation and lactation until weaning of the pups. The test item was administered as a solution in degassed purified water, by oral gavage, at dose-levels of 10, 20 or 40 mg a.i./kg/day (a.i. = active ingredient). Another group of 25 males and 25 females received the vehicle alone under the same experimental conditions and acted as a control group. A constant dosage‑volume of 5 mL/kg/day was used. The animals were checked at least twice daily for mortality or morbidity and at least once daily for clinical signs. A detailed clinical examination was performed once before the beginning of the treatment period and then once a week. Body weight and food consumption were recorded weekly. The estrous cycles were monitored during the last 3 weeks before mating and during the mating period, which lasted up to 13 days. The females were allowed to litter and rear their progeny until weaning. The pups were regularly weighed throughout the lactation period and observed daily for clinical signs. Physical and reflex development were assessed (pinna unfolding, hair growth, tooth eruption, auditory canal opening, eye opening, surface righting, cliff avoidance and air righting). At the end of the treatment period or prior to premature sacrifice, the F0 animals were blood sampled for analysis of hematology and blood biochemistry parameters, including ß‑hydroxybutyrate and acetoacetate determination. The animals were not fasted before blood collection. After weaning of the pups, the males and females of the F0 generation were sacrificed. Sperm analysis was performed on the first 10 males of the control and high-dose groups (groups 1 and 4) and since no treatment-related effects were observed this was not performed on males of the other groups. A complete macroscopic examination was performed, including counting the number of implantation sites in females, and designated organs were weighed. A microscopic examination was performed on the reproductive organs and macroscopic lesions of all groups and for the control and high-dose groups the heart, kidneys and liver were also examined. The liver of all intermediate-dose group animals was also examined.

Of the progeny of the F0 generation, one or two males and one or two females per litter were selected to constitute the F1 generation of groups of 25 male and 25 female rats in the control, low and intermediate groups and 27 male and 27 female rats in the high-dose group (this to compensate in advance for mortality around the time of delivery, a known effect of the test item). Three groups received the test item, Sodium thioglycolate(batch No.26461), daily for 10 weeks prior to mating, during mating, gestation and lactation until weaning of the pups. Due to the test item inducing known difficulties around the time of delivery, all mated females, including controls, were not treated from day 19 of gestation until day 1 of lactation in order to see if this would reduce the mortality. The test item was administered as a solution in degassed purified water, by oral gavage, at dose‑levels of 10, 20 or 40 mg a.i./kg/day. Another group of 25 males and 25 females received the vehicle alone under the same experimental conditions and acted as a control group. A constant dosage‑volume of 5 mL/kg/day was used. The animals were checked at least twice daily for mortality or morbidity and at least once daily for clinical signs. A detailed clinical examination was performed once at weaning before the beginning of the treatment period and then once a week. Body weight and food consumption were recorded weekly. Each animal was assessed for sexual maturity (balanopreputial separation or vaginal opening) and the day of age and body weight were recorded on the day each animal was positive. At 4 weeks of age, the animals were assessed for auditory function (acoustic startle response) and pupil constriction, andweeks of age the spontaneous locomotor activity was measured using an automated infra-red sensor equipment. The estrous cycles were monitored during the last 3 weeks before mating and during the mating period, which lasted up to 19 days. The females were allowed to litter and rear their progeny until weaning. The pups were regularly weighed throughout the lactation period and observed daily for clinical signs. Physical and reflex development were assessed (pinna unfolding, hair growth, tooth eruption, auditory canal opening, eye opening, surface righting, cliff avoidance and air righting). In addition, the anogenital distance of all pups was measured on the day after birth and the presence of nipples was checked in all males on post-natal day 12 or 13. At weaning of the pups, the males and females of the F1 generation were sacrificed. Sperm analysis was performed on the first ten males of the control and high-dose groups (groups 1 and 4). A complete macroscopic examination was performed, including counting the number of implantation sites in females, and designated organs were weighed. A microscopic examination was performed on the reproductive organs and any macroscopic lesions of all groups. In addition, pups of both the F0 and F1 animals were submitted for a macroscopic examination. One randomly selected pup/sex/litter for F1 and F2 litters, all pups found dead or prematurely sacrificed and any pups showing external abnormalities or clinical signs were weighed and then submitted for a macroscopic examination of the principal thoracic and abdominal organs with special attention paid to the reproductive organs.

There were no effects of treatment at 10 or 20 mg a.i./kg/day.

At 40 mg a.i./kg/day, the males and females showed no effects of treatment during the pre-mating, mating or gestation phases. At delivery, four females were found dead; one on gestation day 21 and three on gestation day 22. One of these females found dead on gestation day 22 had delivered 12 live and one dead pup before dying, the other three females had not started delivery and had dead fetuses in the uterine horns at necropsy. On lactation day 1, one female, showing signs of poor condition (piloerection, blood and placentae in the bedding), cannibalized her 10 pups (it is likely that more pups were born and cannibalized before being noticed because there were five more implantation sites in the uterine horns than pups). It is not known whether the pups were alive or dead prior to cannibalism, however the adverse outcome is considered to be a maternal effect since no fetuses remained in the uterine horns at necropsy; the female did deliver all pups starting delivery on gestation day 21. No effects of treatment were observed in the F0 generation animals during the remainder of the lactation period other than a slightly lower mean body weight gain of the females during the first 4 days of lactation. There were no effects on sperm parameters in the control or high-dose group males.

There were no treatment-related effects on organ weights at any dose-level. There were no treatment-related microscopic changes in testis, epididymis, prostate, coagulating glands or seminal vesicles. There were possible treatment-related changes in the uterus of one 40 mg a.i./kg/day female (female T20412 which had only implantation scars thought to be left from early resorptions); thrombosis of the mesometrial remnant, organized by granulation tissue composed of fibroplasia, fibrin plugs, thrombi and neovascularization. Golden pigment-laden cells and pyknotic trophoblastic cells were observed. The found dead females had no microscopic findings which could explain the deaths but one female had hemorrhage of a mesometrial triangle. Minimal to moderate periportal hepatocellular microvacuolation was observed at 40 mg a.i./kg/day in 2/25 males and 6/25 females, and in 4/6 prematurely sacrificed/found dead females suggesting mild liver toxicity at this dose-level. No control animals had the same finding.

Female plasma fatty acid concentration was statistically significantly decreased however there were no effects of treatment with the test item on plasma acetoacetate or ß-hydroxybutyrate concentrations indicating that the animals were not in ketosis.

 

There were no effects of treatment on F1 pups in the 10 or 20 mg a.i./kg/day groups.

At 40 mg a.i./kg/day, the pups had a 9.6% mortality rate during the first 4 days of lactation (cannibalism and being found dead); neither the pups nor the F0 dams showed particular clinical signs (except female T20401 which had piloerection and cannibalized all pups in the litter). There was a possible, very slight, delay in physical development; the majority of the pups achieved tooth eruption, eye opening and auditory canal opening on the same day as the majority of the pups from the other groups but there was a higher percentage of pups achieving these landmarks on later days than in the other groups. The females of the F1 generation started treatment (on day 22 of age) with a statistically significantly lower mean body weight than the controls. Mean body weight gain and mean food consumption were both also statistically significantly lower for the first week of treatment. During gestation, the females had a slightly lower body weight gain. Treatment was stopped on gestation day 19 and delivery passed without problem in all females. However, two females were prematurely sacrificed on day 2 or day 5 of lactation due to death of the litter. One of the females had thrombosis of mesometrial triangles. Treatment was re-started on lactation day 1 and the number of pup deaths was higher when compared with the controls. This was mainly due to one female which had three found dead pups and cannibalized 13 pups over several days until lactation day 5 when all pups were dead. Another female also had total litter death but delivered only one pup. The total number of dead pups in this group was similar to that of the controls when the female with the 16 dead pups is excluded (15 dead pupsvs.the control group), but whereas the control pup deaths were concentrated in four litters, at 40 mg a.i./kg/day the pup deaths were spread over seven litters. The females had not had any noticeable problems during delivery and were not showing any clinical signs during the first days of lactation except for a possible effect on maternal nesting and nursing behavior since the pups were often observed to be cold, even those that survived. There were no treatment-related effects on organ weights at any dose-level. There were no effects on sperm parameters in the control or high-dose group males. There were no treatment-related microscopic changes in testis, epididymis, prostate, coagulating glands or seminal vesicles of the males or in the ovaries, oviducts, uterus and vagina of the 40 mg a.i./kg/day females.

The female F2 pups had a slightly lower mean body weight at the end of lactation (-5%) but there were no effects on male or female F2 pup physical development in terms of eye opening, tooth eruption or auditory canal opening or on reflex development.

At 40 mg a.i./kg/day, it is concluded that the test item has no effect on non-pregnant, naïve, adult rats but that it causes maternal toxicity and death of susceptible pregnant females around the time of delivery. Effects on the dam include lack of nesting/nursing behavior and this causes death of the pups which have been delivered. If treatment is stopped just prior to delivery, the females may survive delivery but pup death may still occur and pup clinical signs of coldness suggest that maternal nesting/nursing behavior is still impaired by treatment with the test item and affect pup survival. There is evidence that female rats are more affected by test item treatment than males as shown by lower F1 female body weight and body weight gain. Minimal to moderate periportal heptocellular microvacuolation was observed in females and some male F0 animals treated at 40 mg a.i./kg/day suggestive of mild hepatotoxicity and especially in dams (4/6)found dead or prematurely sacrificed at time of parturition. Sodium thioglycolate is known to induce fatty liver via aninhibition of the β‑oxidation of fatty acids.

There were no effects on sperm parameters in the control or high-dose group males.

There were no effects of treatment on any parameters measured in either males or females with the test item at 10 or 20 mg a.i./kg/day.

 

Under the experimental conditions of this study, and in view of the maternal mortalities and liver effects in males and females observed at 40 mg a.i./kg/day, the dose-level of 20 mg a.i./kg/day was considered to be the No Observed Effect Level (NOEL) for parental toxicity, female fertility and gestation of each generation and for development, growth and survival of the progeny. It is probable that the small effects observed on pup survival at 40 mg a.i./kg/day were secondary to the severe and lethal effects observed in the pregnant dams at that dose level.

 

The No Observed Effect Level (NOEL) for males fertility and female mating behaviour was higher or equal than 40 mg a.i./kg/day.

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
complete and valid
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

2-Generation Study (OECD 416)

The effects of NaTG on the integrity and performance of the male and female reproductive systems, including gonadal function, the estrous cycle, mating behavior, conception, gestation, parturition, lactation and weaning, and on the growth and development of the offspring was evaluated over 2 generations (Davies, OECD 416).

Three groups of 25 male and 25 female Sprague-Dawley rats received NaTG daily for 10 weeks prior to mating, during mating, gestation and lactation until weaning of the pups. NaTG was administered by oral gavage at dose-levels of 10, 20 or 40 mg/kg/day. Another group of 25 males and 25 females received the vehicle alone under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg/day was used. The animals were checked at least twice daily for mortality or morbidity and at least once daily for clinical signs. A detailed clinical examination was performed once before the beginning of the treatment period and then once a week. Body weight and food consumption were recorded weekly. The estrous cycles were monitored during the last 3 weeks before mating and during the mating period, which lasted up to 13 days. The females were allowed to litter and rear their progeny until weaning. The pups were regularly weighed throughout the lactation period and observed daily for clinical signs. Physical and reflex development was assessed (pinna unfolding, hair growth, tooth eruption, auditory canal opening, eye opening, surface righting, cliff avoidance and air righting). At the end of the treatment period or prior to premature sacrifice, the F0 animals were blood sampled for analysis of hematology and blood biochemistry parameters, including ß‑hydroxybutyrate and acetoacetate determination. The animals were not fasted before blood collection. After weaning of the pups, the males and females of the F0 generation were sacrificed. Sperm analysis was performed on the first 10 males of the control and high-dose groups (groups 1 and 4) and since no treatment-related effects were observed this was not performed on males of the other groups. A complete macroscopic examination was performed, including counting the number of implantation sites in females, and designated organs were weighed. A microscopic examination was performed on the reproductive organs and macroscopic lesions of all groups and for the control and high-dose groups the heart, kidneys and liver were also examined. The liver of all intermediate-dose group animals was also examined. Of the progeny of the F0 generation, one or two males and one or two females per litter were selected to constitute the F1 generation of groups of 25 male and 25 female rats in the control, low and intermediate groups and 27 male and 27 female rats in the high-dose group (this to compensate in advance for mortality around the time of delivery, a known effect of the test item). Three groups received NaTG daily for 10 weeks prior to mating, during mating, gestation and lactation until weaning of the pups. Due to the test item inducing known difficulties around the time of delivery, all mated females, including controls, were not treated from day 19 of gestation until day 1 of lactation in order to see if this would reduce the mortality. The test item was administered by oral gavage at dose levels of 10, 20 or 40 mg/kg/day. Another group of 25 males and 25 females received the vehicle alone under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg/day was used. The animals were checked at least twice daily for mortality or morbidity and at least once daily for clinical signs. A detailed clinical examination was performed once at weaning before the beginning of the treatment period and then once a week. Body weight and food consumption were recorded weekly. Each animal was assessed for sexual maturity (balanopreputial separation or vaginal opening) and the day of age and body weight was recorded on the day each animal was positive. At 4 weeks of age, the animals were assessed for auditory function (acoustic startle response) and pupil constriction, and weeks of age the spontaneous locomotor activity was measured using automated infra-red sensor equipment. The estrous cycles were monitored during the last 3 weeks before mating and during the mating period, which lasted up to 19 days. The females were allowed to litter and rear their progeny until weaning. The pups were regularly weighed throughout the lactation period and observed daily for clinical signs. Physical and reflex development was assessed (pinna unfolding, hair growth, tooth eruption, auditory canal opening, eye opening, surface righting, cliff avoidance and air righting). In addition, the anogenital distance of all pups was measured on the day after birth and the presence of nipples was checked in all males on post-natal day 12 or 13. At weaning of the pups, the males and females of the F1 generation were sacrificed. Sperm analysis was performed on the first ten males of the control and high-dose groups (groups 1 and 4). A complete macroscopic examination was performed, including counting the number of implantation sites in females, and designated organs were weighed. A microscopic examination was performed on the reproductive organs and any macroscopic lesions of all groups. In addition, pups of both the F0 and F1 animals were submitted for a macroscopic examination. One randomly selected pup/sex/litter for F1 and F2 litters, all pups found dead or prematurely sacrificed and any pups showing external abnormalities or clinical signs were weighed and then submitted for a macroscopic examination of the principal thoracic and abdominal organs with special attention paid to the reproductive organs.

There were no effects of treatment at 10 or 20 mg/kg/day.

At 40 mg/kg/day, the males and females showed no effects of treatment during the pre-mating, mating or gestation phases. At delivery, four females were found dead; one on gestation day 21 and three on gestation day 22. One of these females found dead on gestation day 22 had delivered 12 live and one dead pup before dying; the other three females had not started delivery and had dead fetuses in the uterine horns at necropsy. On lactation day 1, one female, showing signs of poor condition (piloerection, blood and placentae in the bedding), cannibalized her 10 pups (it is likely that more pups were born and cannibalized before being noticed because there were five more implantation sites in the uterine horns than pups). It is not known whether the pups were alive or dead prior to cannibalism, however the adverse outcome is considered to be a maternal effect since no fetuses remained in the uterine horns at necropsy; the female did deliver all pups starting delivery on gestation day 21. No effects of treatment were observed in the F0 generation animals during the remainder of the lactation period other than a slightly lower mean body weight gain of the females during the first 4 days of lactation. There were no effects on sperm parameters in the control or high-dose group males. There were no treatment-related effects on organ weights at any dose-level. There were no treatment-related microscopic changes in testis, epididymis, prostate, coagulating glands or seminal vesicles. There were possible treatment-related changes in the uterus of one 40 mg/kg/day female (one female which had only implantation scars thought to be left from early resorptions); thrombosis of the mesometrial remnant, organized by granulation tissue composed of fibroplasia, fibrin plugs, thrombi and neovascularization. Golden pigment-laden cells and pyknotic trophoblastic cells were observed. Female had hemorrhage of a mesometrial triangle. Minimal to moderate periportal hepatocellular microvacuolation was observed at 40 mg/kg/day in 2/25 males and 6/25 females, and in 4/6 prematurely sacrificed/found dead females suggesting mild liver toxicity at this dose-level. No control animals had the same finding.

Female plasma fatty acid concentration was statistically significantly decreased however there were no effects of treatment with the test item on plasma acetoacetate or ß-hydroxybutyrate concentrations indicating that the animals were not in ketosis.

There were no effects of treatment on F1 pups in the 10 or 20 mg/kg/day groups.

At 40 mg/kg/day, the pups had a 9.6% mortality rate during the first 4 days of lactation (cannibalism and being found dead); neither the pups nor the F0 dams showed particular clinical signs (except one female which had piloerection and cannibalized all pups in the litter). There was a possible, very slight, delay in physical development; the majority of the pups achieved tooth eruption, eye opening and auditory canal opening on the same day as the majority of the pups from the other groups but there was a higher percentage of pups achieving these landmarks on later days than in the other groups. The females of the F1 generation started treatment (on day 22 of age) with a statistically significantly lower mean body weight than the controls. Mean body weight gain and mean food consumption were both also statistically significantly lower for the first week of treatment. During gestation, the females had a slightly lower body weight gain. Treatment was stopped on gestation day 19 and delivery passed without problem in all females. However, two females were prematurely sacrificed on day 2 or day 5 of lactation due to death of the litter. One of the females had thrombosis of mesometrial triangles. Treatment was re-started on lactation day 1 and the number of pup deaths was higher when compared with the controls. This was mainly due to one female which had three found dead pups and cannibalized 13 pups over several days until lactation day 5 when all pups were dead. Another female also had total litter death but delivered only one pup. The total number of dead pups in this group was similar to that of the controls when the female with the 16 dead pups is excluded (15 dead pupsvs.control group), but whereas the control pup deaths were concentrated in four litters, at 40 mg/kg/day the pup deaths were spread over seven litters. The females had not had any noticeable problems during delivery and were not showing any clinical signs during the first days of lactation except for a possible effect on maternal nesting and nursing behavior since the pups were often observed to be cold, even those that survived. There were no treatment-related effects on organ weights at any dose-level. There were no effects on sperm parameters in the control or high-dose group males. There were no treatment-related microscopic changes neither in testis, epididymis, prostate, coagulating glands or seminal vesicles of the males nor in the ovaries, oviducts, uterus and vagina of the 40 mg/kg/day of the females.

The female F2 pups had a slightly lower mean body weight at the end of lactation (-5%) but there were no effects on male or female F2 pup physical development in terms of eye opening, tooth eruption or auditory canal opening or on reflex development.

At 40 mg/kg/day, it is concluded that the test item has no effect on non-pregnant, naïve, adult rats but that it causes maternal toxicity and death of susceptible pregnant females around the time of delivery. Effects on the dam include lack of nesting/nursing behavior and this causes death of the pups which have been delivered. If treatment is stopped just prior to delivery, the females may survive delivery but pup death may still occur and pup clinical signs of coldness suggest that maternal nesting/nursing behavior is still impaired by treatment with the test item and affect pup survival. There is evidence that female rats are more affected by test item treatment than males as shown by lower F1 female body weight and body weight gain.

Minimal to moderate periportal heptocellular microvacuolation was observed in females and some male F0 animals treated at 40 mg/kg/day suggestive of mild hepatotoxicity and especially in dams (4/6) found dead or prematurely sacrificed at time of parturition. NaTG is known to induce fatty liver via an inhibition of theβ-oxidation of fatty acids.

There were no effects on sperm parameters in the control or high-dose group males.

There were no effects of treatment on any parameters measured in either males or females with the test item at 10 or 20 mg/kg/day.

Under the experimental conditions of this study, and in view of the maternal mortalities and liver effects in males and females observed at 40 mg/kg/day, the dose-level of 20 mg/kg/day was considered to be the No Observed Effect Level (NOEL) for parental toxicity, female fertility and gestation of each generation and for development, growth and survival of the progeny. It is probable that the small effects observed on pup survival at 40 mg/kg/day was secondary to the severe and lethal effects observed in the pregnant dams at that dose level. The No Observed Effect Level (NOEL) for males fertility and female mating behavior was higher or equal than 40 mg/kg/day.

 

Reproduction/Developmental Screening Study (OECD 421)

In a reproduction/developmental screening test performed according to the OECD Guideline 421, four groups of 12 male and 12 female Sprague-Dawley rats received sodium thioglycolate (purity 98.9% pure), daily, by oral (gavage) administration, 10 weeks before mating and through mating and, for the females, through gestation until day 5 post-partum at dose-levels of 0, 20, 40 or 80 mg/kg bw/d.

Clinical signs and mortality were checked daily. Body weight and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. The animals were paired for mating and the dams were allowed to litter and rear their progeny until day 5 post-partum. The total litter sizes and numbers of pups of each sex were recorded after birth, pup clinical signs were recorded daily and pup body weights were recorded on days 1 and 5 post-partum. The males were sacrificed after completion of the mating period and the females on day 5 post-partum (or on day 25 post-coitum for females which did not deliver). The body weight and selected organs (brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles and testes and uterus) were weighed and a macroscopic post-mortem examination of the principal thoracic and abdominal organs and a microscopic examination of selected organs (macroscopic lesions, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles, testes, and uterus) were performed. In the females, which were apparently non-pregnant, the presence of implantation scars on the uterus was checked using ammonium sulphide staining technique. Epididymal sperm was sampled for motility, morphology and count and testicular sperm heads resistant to homogenization (i.e. elongated spermatids and mature spermatozoa) were counted. The pups were sacrificed on day 5 post-partum and were carefully examined for gross external abnormalities and a macroscopic post-mortem examination was performed.

Two males and one female given 80 mg/kg bw/d were found dead during the pre-mating or mating periods with no clinical signs observed before death and no relevant post-mortem findings. Four females at 80 mg/kg/d and one at 40 mg/kg bw/d were found dead or were prematurely sacrificed because of difficulties to deliver. Ptyalism was observed at 40 and 80 mg/kg bw/d with a dose-related incidence and may be related to the taste of dosing solution. All male and female groups had body weight gains comparable with the controls throughout the study. There were no adverse effects of treatment on mean food consumption, except a slight lowering of food consumption during the lactation period for females given 80 mg/kg bw/d.

The mean number of estrous cycles in each group was between 4 and 5 during the period measured and the mean cycle length was a normal 4 or 5 days. All pairs mated and the majority of the females were pregnant. There were no effects of treatment on the mean number of days taken to mate.

Females given 80 mg/kg bw/d had a statistically significantly longer gestation period, a non-statistically significantly lower number of corpora lutea and a statistically significantly lower number of implantations and pups. One female had total resorptions and one litter died on day 1 post-partum.

There were no effects of treatment on sperm morphology, motility or counts. The mean absolute seminal vesicle weights were statistically significantly lower for all treated groups compared to the control group, but the mean relative weight was only decreased at 80 mg/kg bw/d. For all treated groups, the absolute weights were in the range of the historical control data. Only at 80 mg/kg bw/d, the relative weight was outside the historical data and was correlated with a slight decrease in secretory content in the seminal vesicles. There were no treatment-related adverse effects in pups based on clinical signs, mean body weight gain and necropsy findings.

The NOAEL for parental toxicity was considered to be 20 mg/kg bw/d (based on deaths at 40 and 80 mg/kg bw/d), the NOAEL for reproductive performance (mating, fertility and delivery) was considered to be 20 mg/kg bw/d (based on deaths at 40 and 80 mg/kg bw/d) and the NOAEL for toxic effects on progeny was 40 mg/kg bw/d (based on the dead litter at 80 mg/kg bw/d which cannot definitively be attributed to maternal condition).

 


Short description of key information:
Thioglycolic acid and its salts are not considered to be reproductive toxicants. The conducted reproduction toxicity studies with sodium thioglycolate can be bridged to other salts of the thioglycolic acid, because in aquous solutions only the organic thioglycolate anion may have the potential to cause reproduction toxicity.

Effects on developmental toxicity

Description of key information
The developmental toxicity of ammonium and sodium thioglycolates has been investigated in standard oral and dermal studies in rats and/or rabbits compliant or comparable to OECD guideline 414, respectively.
When ammonium thioglycolate was administered by gavage, the NOAELs for maternal and embryo-foetal toxicity were 15 and 75 mg/kg bw/d, respectively. No teratogenic effects were observed in all studies.
In the dermal studies with sodium thioglycolate, the NOAELs for maternal toxicity was < 50 mg/kg bw/d in Sprague-Dawley rats and > 65 mg/kg bw/d in New Zealand White rabbits. The developmental toxicity NOAEL was 100 mg/kg bw/d and >65 mg/kg bw/d, respectively.
Link to relevant study records

Referenceopen allclose all

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
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Strain of rats: Hsd/Cpb: WU
Source: Fa. Harlan Winkelmann GmbH, 33178 Borchert
Age: 9-10 weeks old
Acclimatization period: 6 days
Weight range at study initiation: 177-213 g Diet: Altromin Standard Diet TPF N 1234, ad libitum
Water: Tap water, ad libitum

Husbandry:
Housing: Makrolon type III cages
Illumination: artificial lighting from 6.00 a.m. - 6.00 p.m.
Temperature: 21-23.5°C
Relative humidity: 51-66%Vehicle: demineralized water
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- Rate of solution preparation: daily
- Amount of vehicle: 10 ml/kg (base on weight on GD6)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration and homogeneity of ammonium thioglycolate in preparations administred were determined once during the study, on the second day of treatment. The results showed values ranging from 96.5 % to 100 % of the nominal concentrations
Details on mating procedure:
- Groups of four sexually matures, virgin females were left over night with one stock stud.
- Proof of pregnancy: sperm in vaginal smear (GD 0)
Duration of treatment / exposure:
gestation days 6 - 19
Frequency of treatment:
daily
Duration of test:
Duration of test: 15 days - dosing GD 6 -19, animals were killed on day 20
Dose / conc.:
3 mg/kg bw/day
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
75 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Maternal examinations:
Clinical signs including mortality and evidence of abortion were checked daily.
Body weight was determined on GD0, 6 and daily until GD20.
Food and water consumption were recorded at designated intervals during pregnancy.
On day 20 of pregnancy, females were killed. The terminal body weights of the dams were recorded. Females were examined macroscopically.

Litter parameters were recorded: number of corpora lutea, implantation sites, early and late resorptions, and dead and live foetuses.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes:
Fetal examinations:
Foetuses were weighed, sexed, and submittedto external examination. About 1/2 of them were examined for skeletal malformations and 1/2 for organ malformations.The foetus sex was determined on the base of measurment of anogenital indice and by inspection of gonads.
Statistics:
yes: Dunnett test or Fischer-Pitman permutation test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see below
Mortality:
mortality observed, treatment-related
Description (incidence):
see below
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
Rooting in the bedding material was seen in all rats in the high dose group. Two animals in the high dose group died on GD 20, one day after the last administration of the test material. These deaths are regarded to be treatment related.
18 to 24 rats out of 25 per group proved to be pregnant.
The body weight development, food and water consumption were not affected by treatment. The terminal body weights were similar in all groups.
The uterus weights in the high dose group were slightly, but not significantly lower than in the control or the other groups. This finding is regarded to be accidental. The numbers of corpora lutea, implantations and live foetuses were not affected.
The numbers of dead foetuses, complete, early and late resorptions were not increased. The sex distribution was not affected in any of the groups.
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
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
Key result
Dose descriptor:
LOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
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:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
The weights of the foetuses in the low, mid, and high dose groups were similar to the control and not affected by treatment. The frequency of all malformations was in a normal range and not increased.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 75 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
Key result
Dose descriptor:
LOAEL
Effect level:
> 75 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
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
The doses of 3 and 15 mg ammonium thioglycolate/kg were systemically tolerated by the rats.
At dose level of 75 mg/kg, two dams died on GD 20. Rooting in the bedding material had been seen, but no maternotoxic effects.
The no observed effect level (NOEL) for embryo-fetal toxicity was 75 mg/kg.
No teratogenic effects were observed.
Executive summary:

The potential of ammonium mercaptoacetate to induce developmental toxicity after maternal exposure during the critical period of organogenesis was evaluated in rat according to OECD Guideline N° 414 (22nd January 2001) and in compliance with Good Laboratory Practices.

Ammonium mercaptoacetate was administered orally by gavage to four groups of 25 bred female Sprague-Dawley rats once daily from gestation days 6 through 19. Dosage levels were 0, 3, 15, and 75 mg/kg/d.

The fetuses were delivered by caesarean section on GD 20 and examined for macroscopic malformations. About 1/2 of them were examined for skeletal malformations and 1/2 for organ malformations. Clinical signs including mortality and evidence of abortion were checked daily. Body weight was determined on GD0, 6 and the daily until GD20. Food and water consumption was recorded at designated intervals during pregnancy. On day 20 of pregnancy, females were killed. The terminal body weights of the dams were recorded. The gravid uterus was weighed and foetuses removed by hysterectormy. Females were examined macroscopically.

The doses of 3 and 15 mg ammonium thioglycolate/kg were systemically tolerated by the rats. All animals in the low and medium level dose group survived to the scheduled necropsy. At the dose level of 75 mg/kg, two dams died on GD20, one day after the last administration of test material. These death were considered treatment related. At this dose level, rooting in the bedding material had been seen in all rats, but no maternotoxic effects. No treatment related internal findings were observed at any dose level. No clinical signs that could be attributed to ammonium mercaptoacetate were observed in the treated groups. Body weight and food consumption were not affected by treatment. Intrauterine growth and survival were unaffected by ammonium mercaptoacetate administration at all dose levels. Any significant or relevant foetal external, soft tissue and skeletal malformations were observed at any dose level. The developmental variations expressed in the treated groups were generally similar to those present in the control group or occurred in a manner that was not dose related. No teratogenic effects were observed

Under these experimental conditions, the No-Observed-Adverse-Effect-Level (NOAEL) was 15 mg/kg/d for maternal toxicity. A NOAEL has been determined at 75 mg/kg/d for embryo-foetal toxicity.

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
Reference:
Composition 0
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Test animals:        
- Source: Covance Research Products (Denver, PA)         
- Age: no data         
- Body weight at GD 0: 2.-876- 4.327 kg
- Acclimatation period : 3 days         
- Diet: Purina Certified Rabbit Chow (No. 5322), ad libitum         
- Water: Tap water, ad libitum        
- Housing: singly housed in stainless-steel cages with mesh flooring.         

Environmental conditions:        
- Temperature: 64.1-74.41F         
- Humidity: 41.6-86.5%         
- Air changes: no data         
- Photoperiod: Alternating 12-hour light and dark cycles
Route of administration:
dermal
Vehicle:
other: 95%  ethanol:distilled water, 1:1
Details on exposure:
0.5 ml/kg of test material in vehicle or vehicle alone was applied  directly to a 3x3 inch shaved area on the dorsum of the rat with a glass  syringe fitted with a 16-gauge, stainless-steel, ball-tipped dosing  needle. The formulation was allowed to remain on the animal for a minimum  of 6 hr/treatment day. At the end of each 6-hr period, the application  site was wiped with warm-water-soaked gauze to remove residual vehicle or  sodium thioglycolate.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prepared weekly based on stability information and were verified to be  within 85.1-111.7% of target.
Duration of treatment / exposure:
gestation days 6-29
Frequency of treatment:
daily
Duration of test:
necropsy on GD 30
Dose / conc.:
10 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)
Dose / conc.:
65 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 (2 replicates of 12)
Control animals:
yes, concurrent vehicle
Details on study design:
Range-finding study: Dose selection in rabbits was based on a topical developmental toxicity  screen in pregnant rabbits. That study indicated that sodium  thioglycolate was maternally lethal (>=50% of the does) at 100, 150, and  175 mg/kg/day. Because of the steep dose-response curve in the screening  study, a dose of 65 mg/kg/day was chosen as the top dose and anticipated  to result in moderate maternal and developmental toxicity. If excessive  toxicity occurred at 65 mg/kg/day, 50 mg/kg/day was also chosen to be  available as a potential high dose for the second replicate, if  necessary. Doses for the 30 and 50 mg/kg/day groups, however, were  inadvertently formulated as 15 and 25 mg/kg/day (i.e., 50% of the  original target doses) for the first replicate. Because of the  dose-related increase in erythema and oedema at the dosing site seen at  these lower doses, the second replicate also employed doses of 0, 10, 15,  25, and 65 mg/kg/day.
Maternal examinations:
Twelve naturally mated females were assigned to each group in a two replicate design for a total of twenty-four does/group and monitored at regular intervals throughout gestation for clinical signs (including  dosing site condition), feed intake, and body weight.
At necropsy on  gestational day 30,  maternal clinical  condition; body, liver, kidney (paired) weights were recorded.
Ovaries and uterine content:
At necropsy on  gestational day 30, gravid uterine weights; and  pregnancy status were recorded:. The number of ovarian corpora lutea and uterine  implantations (resorbed, dead, or live foetuses) was recorded. 
Fetal examinations:
All live  foetuses were counted, weighed, and examined for external alterations,  including cleft palate. One hundred percent of the live foetuses per  litter were sexed internally and examined for visceral alterations.  Approximately 50% of the foetuses were decapitated and the heads fixed,  decalcified, and examined for soft tissue craniofacial alterations.  All  foetal carcasses were eviscerated, macerated, and stained with alizarin  red S and alcian blue. All foetuses were examined for ossified and  cartilaginous skeletal alterations.
Statistics:
The unit for statistical measurement was the pregnant female or the  litter. Quantitative continuous data (e.g., maternal body weights, foetal  body weights, feed consumption, etc.) were compared among treatment  groups, per species, by parametric statistical tests whenever Bartlett  test for homogeneity of variance was not significant.  General linear models (GLM) procedures were applied to the ANOVA and the  tests for linear trend. Before GLM analysis, an arc sine-square root  transformation was carried out on all litter-derived percentage data  (e.g., % resorptions/litter, % malformations/litter, % variations/litter)  (Snedecor and Cochran, 1967). For litter-derived percentage data, the  ANOVA was weighted according to litter size. When a significant (p <  0.05) main effect for dose occurred, Dunnett's multiple comparison test  (Dunnett, 1955, 1964) was used to compare each treatment group to the  control group for that measure. A one-tailed (i.e., Dunnett's test) was used for all pairwise comparisons  to the vehicle control group, except that a two-tailed test was used for  maternal body and organ weight parameters, maternal feed consumption,  fetal body weight, and percent males per litter. Data for any measure that showed a significant (p < 0.05) dose X  replicate interaction in a two-way (dose X replicate) ANOVA were further  evaluated for dose effects within each replicate using a one-way ANOVA,  test for linear trend, and Dunnett's test. The data are presented pooled  across the two replicates for the rabbit. All nominal scale measures were analyzed by w2 for Independence for  differences among treatment groups (Snedecor and Cochran, 1967) and by  the Cochran-Armitage test for linear trend on proportions (Cochran, 1954;  Armitage, 1955; Agresti, 1990). The alpha level for each statistical  comparison was 0.05, and the significance levels for trend tests and  pairwise comparisons were reported as p < 0.05 or p < 0.01. necropsy.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
effects observed, treatment-related
Description (incidence and severity):
see below
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
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
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
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
No maternal deaths were associated with sodium thioglycolate treatment.
One female in the 25 mg/kg per day group delivered early and one doe in the 65 mg/kg per day group were removed due to a pre-existing condition (trichobezoar or hairball in the stomach).
Clinical observations were almost exclusively limited to effects of treatment on the dosing site. Very slight erythema in few females during the first two days of dosing at greater than or equal to 15 mg/kg per day occurred and progressed to include does at 10 mg/kg per day after 4 days of dosing. Moderate to severe erythema and oedema occurred at doses greater than or equal to 15 mg/kg per day and oedema occurred in all dose groups by gestational day 16. Petechia at the dosing site and dry skin also was noted in the high dose group as early as gestational day 9 but was also seen in one doe at 25 mg/kg per day.
Maternal body weight and body weight gain were equivalent across dose groups for all intervals measured except for body weight gain for gestational days 12-15. There was a trend for decreased body weight gain for this interval, which was significant in the high dose group when compared to controls.
Absolute feed consumption per rabbit (grams per day) was significantly reduced from gestational days 9-12 in the 10 mg/kg per day group (Replicate II only) and for gestational days 15-18 at 65 mg/kg per day (Replicate II). Relative feed consumption (grams per kilogram per day) was significantly decreased for gestational days 6-9 (65 mg/kg per day, Replicate II), gestational days 9-12 (10 and 65 mg/kg per day, Replicate II), gestational day 12-15 at 15 mg/kg per day (Replicate I), gestational days 15- 18 for 10 and 65 mg/kg per day (Replicate II), and 18-21 at 25 mg/kg per day (Replicate II). Maternal feed consumption in grams per day as well as in grams per kg per day for gestational days 29-30 was significantly reduced at 15 mg/kg per day for Replicate I only. Therefore, there were no consistent treatment related reductions in feed consumption.
At scheduled necropsy, there was no effect of treatment on terminal maternal body weight. In addition, organ weights and gravid uterine weights were equivalent across groups.
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
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
>= 65 mg/kg bw/day
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
LOAEL
Effect level:
> 65 mg/kg bw/day
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
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:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Prenatal viability was unaffected by maternal exposure to sodium thioglycolate. The incidences of foetal external, visceral, and skeletal alterations were also unaffected. Body weights of male and female foetuses per litter and percent males and females per litter were equivalent across dose groups.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 65 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
Key result
Dose descriptor:
LOAEL
Effect level:
> 65 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
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Topically applied sodium thioglycolate resulted in toxicity to the doe at all dose groups but only at the dosing site; no systemic toxicity was seen at any dose group. There was no evidence of embryo/foetal toxicity in any dose group or treatment related teratogenicity. The maternal NOAEL for systemic toxicity was at or above 65 mg/kg/day; for localized toxicity at the dosing site, it was below 10 mg/kg/day. The NOAEL for developmental toxicity was >65 mg/kg/day.
Executive summary:

The developmental toxicity of sodium thioglycolate was evaluated in pregnant rabbits in a study performed according to a method comparable to the OECD guideline # 414. Pregnant New Zealand White Rabbits were exposed topically, 6 hr/day, to sodium thioglycolate (99% pure) in vehicle (95% ethanol:distilled water, 1:1) from gestational day (GD) 6 through 29 at dose levels of 0, 10, 15, 25 or 65 mg/kgbw/d.

Twenty-four naturally mated female rabbits (in two replicates of 12) were assigned to each group and monitored at regular intervals throughout gestation for clinical signs (including dosing site condition), feed and water intake, and body weight. At necropsy on gestational day 30; the following were recorded: maternal clinical condition; body, liver, kidney and gravid uterine weights; and pregnancy status.The number of ovarian corpora lutea and uterine implantations (resorbed, dead, or live foetuses) was recorded. All live foetuses were counted, weighed, and examined for external alterations, including cleft palate. 1eads fixed, decalcified, and examined for soft tissue craniofacial alterations. All foetal carcasses were eviscerated (and rat foetuses not scheduled for a visceral examination were sexed internally), macerated, and stained with alizarin red S and alcian blue. Intact rat foetuses (those not decapitated) and all rabbit foetuses were examined for ossified and cartilaginous skeletal alterations.

In rabbits, no maternal deaths were associated with sodium thioglycolate treatment. One female in the 25 mg/kg bw/d group delivered early and one doe in the 65 mg/kg bw/d group was removed due to a pre-existing condition (trichobezoar or hairball in the stomach). Clinical observations were almost exclusively limited to effects of treatment at the dosing site (skin erythema) in all groups.

Maternal body weight and body weight gain were equivalent across dose groups for all intervals measured except for body weight gain for gestational days 12-15. There was a trend for decreased body weight gain for this interval, which was significant in the high dose group when compared to controls. There were no consistent treatment related reductions in feed consumption. At scheduled necropsy, there was no effect of treatment on terminal maternal body weight. In addition, organ weights and gravid uterine weights were equivalent across groups.

Prenatal viability was unaffected by maternal exposure to sodium thioglycolate. The incidences of foetal external, visceral, and skeletal alterations were also unaffected. Body weights of male and female foetuses per litter and percent males and females per litter were equivalent across dose groups.

The maternal NOAEL for systemic toxicity was at or above 65 mg/kg bw/d; for local toxicity at the dosing site, it was below 10 mg/kg bw/d. The NOAEL for developmental toxicity was at or above 65 mg/kg bw/d.

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
Reference:
Composition 0
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Test animals:         
- Source: Charles River Laboratories (Raleigh, NC).        
- Age: no data         
- Weight at dosing: ca. 250 g        
-  Acclimation period: no data        
- Diet: Purina Certified Rodent Chow (No. 5002), ad libitum        
-  Water: Tap water, ad libitum         
- Housing: singly housed in solid-bottom  polycarbonate cages with stainless steel wire lids with Sani-Chips certified hardwood bedding

Environmental conditions:         
- Temperature: 70.0-72.81 F         
- Humidity: 28.0-58 %         
- Air changes: no data         
- Photoperiod: Alternating 12-hour light and dark cycles
Route of administration:
dermal
Vehicle:
other: 95% ethanol:distilled water, 1:1
Details on exposure:
Treatment: 1.5 ml/kg of test material in vehicle or vehicle alone was applied  directly to a 3x3 inch shaved area on the dorsum of the rat with a glass  syringe fitted with a 16-gauge, stainless-steel, ball-tipped dosing  needle. The formulation was allowed to remain on the animal for a minimum  of 6 hr/treatment day. At the end of each 6-hr period, the application  site was wiped with warmwater-soaked gauze to remove residual vehicle or  sodium thioglycolate.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prepared weekly based on stability information and were verified to be  within 92.7-103.5 % of target.
Duration of treatment / exposure:
gestation days 6-19
Frequency of treatment:
daily
Duration of test:
necropsy on GD 20
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Pregnant Sprague Dawley derived (CD.) rats were topically exposed to sodium thioglycolate in vehicle (50, 100, and 200 mg/kg/day) or vehicle  alone (95% ethanol:distilled water, 1:1) from gestational day (gd) 6  through 19.
Maternal examinations:
Females were monitored at regular intervals throughout gestation for clinical signs (including dosing site condition), feed and water intake, and body weight. At necropsy on GD 20, the following were recorded: maternal clinical condition; body, liver and gravid uterine weights; and pregnancy status.
Ovaries and uterine content:
The number of ovarian corpora lutea and uterine implantations (resorbed,  dead, or live fetuses) was recorded.
Fetal examinations:
All live foetuses were counted, weighed, and examined for external alterations, including cleft palate. Approximately 50% of the live foetuses per litter were sexed internally  and examined for visceral alterations. These foetuses were decapitated  and the heads fixed, decalcified, and examined for soft tissue  craniofacial alterations. All foetal carcasses were eviscerated (and  foetuses not scheduled for a visceral examination were sexed internally),  macerated, and stained with alizarin red S and alcian blue. Intact  foetuses (those not decapitated) were examined for ossified and  cartilaginous skeletal alterations.
Statistics:
The unit for statistical measurement was the pregnant female or the  litter. Quantitative continuous data (e.g., maternal body weights, foetal  body weights, feed consumption, etc.) were compared among treatment  groups, per species, by parametric statistical tests whenever Bartlett  test for homogeneity of variance was not significant.  General linear models (GLM) procedures were applied to the ANOVA and the  tests for linear trend. Before GLM analysis, an arc sine-square root  transformation was carried out on all litter-derived percentage data  (e.g., % resorptions/litter, % malformations/litter, % variations/litter)  (Snedecor and Cochran, 1967). For litter-derived percentage data, the  ANOVA was weighted according to litter size. When a significant (p <  0.05) main effect for dose occurred, Dunnett's multiple comparison test  (Dunnett, 1955, 1964) was used to compare each treatment group to the  control group for that measure. A one-tailed (i.e., Dunnett's test) was used for all pairwise comparisons  to the vehicle control group, except that a two-tailed test was used for maternal body and organ weight  parameters, maternal feed consumption, fetal body weight, and percent  males per litter.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
effects observed, treatment-related
Description (incidence and severity):
see below
Mortality:
mortality observed, treatment-related
Description (incidence):
see below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see below
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
see below
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
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
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
Sodium thioglycolate treatment was associated with one maternal death at 200 mg/kg/day (only clinical observations at the dosing site preceded death). Other effects, dependent on dose and exposure duration, included body weight and weight gain reductions, changes in relative feed and water intake, and discolouration and slight erythema at the application site. Feed consumption in g/kg/day was significantly increased above the control at 50 (5.4%) and 100 (6.1%) mg/kg/day, but not at 200 mg/kg/day (2.2%). Maternal water consumption in g/kg/day was significantly increased at 200 mg/kg/day, (12.7%) and slightly (but not statistically significantly) increased at 50 (3.2%) and 100 (5.1%) mg/kg/day. Maternal body weights and body weight changes were decreased only at 200 mg/kg/day. At scheduled necropsy, there were no macroscopic indications of organ toxicity. In addition, organ weights and gravid uterine weights were equivalent across groups; maternal body weight was significantly reduced at 200 mg/kg/day.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
< 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
food consumption and compound intake
Key result
Dose descriptor:
LOAEL
Effect level:
<= 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
see below
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Prenatal viability was unaffected by maternal exposure to sodium  thioglycolate. The incidences of foetal external, visceral, and skeletal  alterations were also unaffected. A significant, dose-related, upward  trend was present for % foetuses with skeletal variations per litter, but  there were no significant pairwise comparisons to the vehicle control  group value. The historical data presented in the study report shown a  range of 0.58 to 25.65 % for this parameter, with 9 out of 21 studies  (43%) with a % higher than 15.1% (the highest value observed at 200 mg/kg  bw/d). Accordingly, this effect was not considered as treatment-related.  Body weights of male and female foetuses per litter at 200 mg/kg/day were  significantly lower than control values.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
Key result
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Maternal Toxicity in CD Rats Exposed to Sodium Thioglycolate by Topical Application on Gestational Days 6 Through 19

                                              

Sodium thioglycolate (mg/kg/day, dermal)

0

50

100

200

Maternal pregnancy status

Treated, n

25

25

25

25

Removed, n

0

0

0

0

Dead or euthanized, n

0

0

0

1a

Pregnant at sacrifice, n(%)

25(100)

23(92)

23(92)

21(88)b

Maternal body weight changes (g)c,d

Pretreatment wt. gain (GD 0–6)

32.2±1.3h

29.21±1

28.4±2.1

26.5±1.6

Gestation weight gain (GD 0–20)

135.6±3.3i

129.7±4.4

127.0±5.0

112.5±5.9k

Treatment weight gain (GD 6–20)

110.1±3.1i

106.5±4.0

104.8±3.8

92.7±5.7j

Corrected weight gaine

49.6±1.8i

45.6±2.4

43.9±3.3

34.8±2.8k

Gravid uterine wt.

86.0±3.2

84.1±3.1

83.0±3.1

77.6±4.9

Maternal organ weightsc,f

Liver Absolute (g)

17.78±0.23

17.64±0.40

17.69v0.29

17.02±0.27

Relative (% sacrifice wt.)f

4.53±0.04

4.56±0.07

4.62±0.05

4.65±0.08

Kidney Absolute (g)

2.22±0.04

2.19±0.05

2.26±0.05

2.20±0.04

Relative (% sacrifice wt.)f

0.57±0.01h

0.57±0.01

0.59±0.01

0.60±70.01

Maternal feed consumptionc,g

Pretreatment period (gd 0–6)

Absolute (g/day)

23.4±0.5

23.2±0.6

22.6±0.6

22.5±0.4

Relative (g/kg/day)

85.0±1.4

84.6±1.7

83.3±2.0

83.6±1.3

Treatment period (GD 6–20)

Absolute (g/day)

26.3±0.3

27.5±0.4

27.3±0.5

25.5±0.4

Relative (g/kg/day)

77.1±0.5

81.3±0.9k

81.8±0.8k

78.8±0.8

Maternal water consumptionc,g

Pretreatment period (gd 0–6)

Absolute (g/day)

35.7±1.2

34.2±0.9

32.4±1.1

34.1±0.9

Relative (g/kg/day)

129.8v4.3

125.2v2.9

119.0±3.5

126.9±3.1

Treatment period (gd 6–20)

Absolute (g/day)

43.6±1.3

44.1±1.3

44.4±1.1

46.2±1.2

Relative (g/kg/day)

127.1±3.2i

131.2±3.3

133.6±2.6

143.3v3.9k

aDam 4 was found dead on the morning of gd 18.

bDam 31 had only one implantation site, and no live fetuses on gd 20.

cIncludes all dams pregnant at sacrifice; mean 7 SEM; gd, gestational day.

dBody weights were recorded in the morning of each designated gestational day.

eWeight change during gestation minus gravid uterine weight.

fCalculated using body weight at the time of sacrifice on gd 20.

gRelative feed and water consumption (g/kg/day) were calculated using appropriate body weights.

hp<0.05; Test for linear trend.

ip<0.01; Test for linear trend.

jp<0.05; Dunnett’s test

k p<0.01; Dunnett’s test.

Developmental Toxicity in CDsRat Fetuses Following Maternal Exposure to Sodium Thioglycolate by Topical. Application on Gestational Days 6 Through 19

Sodium Thioglycolate (mg/kg/day, dermal)

0

50

100

200

All littersa,b

25

23

23

21

No. corpora lutea/dam

17.5±0.4

16.7±0.5

16.4±0.5

16.8±0.7

No. implantation sites/litter

15.6±0.5

15.1±0.6

14.7±0.7

15.4±v0.8

% preimplantation loss/litter

11.2±2.7

10.3±2.4

11.6±3.6

10.5±4.1

% resorptions/litter

3.9±1.3

4.6±1.4

3.0±0.9

11.2±5.1

% litters with resorptions

40

48

35

52

% late fetal deaths/litter

0.0±0.0

0.3±0.3

0.0±0.0

0.0±0.0

% litters with late fetal deaths

0

4

0

0

Live littersb,c

25

23

23

20d

No. live fetuses/litter

15.0±0.6

14.4±0.6

14.2±0.6

15.1±0.6

Avg. male fetal body wt./litter (g)

3.73±0.06f

3.70±0.05

3.75±0.08

3.42±0.08g

Avg. female fetal body wt./litter (g)

3.50±0.05e

3.56±0.05

3.61±0.10

3.2570.08g

% male fetuses/litter

45.173.6

52.373.5

50.673.2

52.5±2.3

% externally malformed fetuses/litter

0.0±0.0

0.0±0.0

0.9±0.7

0.3±0.3

% viscerally malformed fetuses/litter

0.0±0.0

0.5±0.5

0.0±0.0

2.2±1.7

% skeletally malformed fetuses/litter

2.3±1.8

2.2±1.0

1.2±0.8

4.5±2.6

% malformed fetuses/litter

1.1±0.9

1.4±0.6

1.5±0.7

3.4±2.2

% fetuses with external variations/litter

0.5±0.5

0.5±0.4

0.0±0.0

0.3±0.3

% fetuses with visceral variations/litter

3.3±1.3

5.0±1.5

3.7±1.7

3.7±2.2

% fetuses with skeletal variations/litter

3.9±1.3f

6.4±2.3

11.2±3.0

15.1±4.2

% fetuses with variations/litter

4.1±1.0

6.3±1.4

7.3±1.7

9.9±2.6

aIncludes all dams pregnant at sacrifice; litter size¼no. implantation sites per dam.

bReported as the mean 7 SEM.

cIncludes only dams with live fetuses; litter size¼no. live fetuses per dam.

dOne female at 200 mg/kg/day had a fully resorbed litter at scheduled necropsy.

ep<0.05; Test for linear trend.

fp<0.01; Test for linear trend.

gp<0.05; Dunnett’s test.

Conclusions:
Topically applied sodium thioglycolate resulted
in toxicity to the dam at 200 mg/kg/day (expressed as
reduced body weights and weight gain, increased relative
water consumption, observations at the dosing site and one
death). Treatment-related increases in feed consumption
(and observations at the application site) were also present
at 50 and 100 mg/kg/day, in the absence of increased body
weights or body weight change. This is an uncommon finding
and has been previously observed in rats exposed to sodium
thioglycolate, attributed to a block in fatty acid
metabolism. Sodium thioglycolate at 200 mg/kg/day also
resulted in significantly decreased male and female foetal
body weights per litter, with no other evidence of
embryofoetal toxicity and no evidence of treatment-related
teratogenicity. Based on the study findings, a no observed
adverse effect level (NOAEL) could not be identified for
maternal toxicity while for developmental toxicity, 100
mg/kg/day was the NOAEL.
Executive summary:

The developmental toxicity of sodium thioglycolate was evaluated in pregnant rats in a study performed according to a method comparable to the OECD guideline # 414. Pregnant Sprague-Dawley rats were exposed topically, 6 hr/day, to sodium thioglycolate (99% pure) in vehicle (95% ethanol:distilled water, 1:1) from gestational day (gd) 6 through 19 at dose levels of 0, 50, 100, and 200 mg/kg bw/d. Twenty-five timed-mated female rats were assigned to each group and monitored at regular intervals throughout gestation for clinical signs (including dosing site condition), feed and water intake, and body weight. At necropsy on gestational day 20; the following were recorded: maternal clinical condition; body, liver and gravid uterine weights; and pregnancy status. The number of ovarian corpora lutea and uterine implantations (resorbed, dead, or live foetuses) was recorded. All live foetuses were counted, weighed, and examined for external alterations, including cleft palate. Approximately 50% of the live rat foetuses per litter were sexed internally and examined for visceral alterations. These foetuses were decapitated and the heads fixed, decalcified, and examined for soft tissue craniofacial alterations. All foetal carcasses were eviscerated (and rat foetuses not scheduled for a visceral examination were sexed internally), macerated, and stained with alizarin red S and alcian blue. Intact rat foetuses (those not decapitated) were examined for ossified and cartilaginous skeletal alterations.

Sodium thioglycolate treatment was associated with one maternal death at 200 mg/kg/d (only clinical observations at the dosing site preceded death). Other effects, dependent on dose and exposure duration, included body weight and weight gain reductions, changes in relative feed and water intake, and discoloration and slight erythema at the application site. Feed consumption was significantly increased above the control at 50 (5.4%) and 100 (6.1%) mg/kg bw/d, but not at 200 mg/kg bw/d (2.2%). Maternal water consumption was significantly increased at 200 mg/kg bw/d (12.7%) and slightly (but not statistically significantly) increased at 50 (3.2%) and 100 (5.1%) mg/kg bw/d. Maternal body weights and body weight changes were decreased only at 200 mg/kg bw/d. At scheduled necropsy, there were no macroscopic indications of organ toxicity. In addition, organ weights and gravid uterine weights were equivalent across groups; maternal body weight was significantly reduced at 200 mg/kg/d. Prenatal viability was unaffected by maternal exposure to sodium thioglycolate. The incidences of foetal external, visceral, and skeletal alterations were also unaffected. A significant, dose-related, upward trend was present for % foetuses with skeletal variations per litter, but there were no significant pairwise comparisons to the vehicle control group value and the % were in the range of the historical control data . Body weights of male and female foetuses per litter at 200 mg/kg bw/d were significantly lower than control values. The maternal NOAEL could not be identified.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
complete and valid
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

The developmental toxicity potential of ammonium thioglycolate by the oral route was evaluated in rats in a study performed according to the OECD Guideline 414 (Gleich, 1998).

Ammonium thioglycolate was administered by gavage to 3 groups of 25 pregnant Wistar rats from gestation days (GD) 6 to 19 at daily doses of 0, 3, 15, and 75 mg/kg bw/d. Day 0 of pregnancy was designated as the day of confirmed mating. Clinical signs including mortality and evidence of abortion were checked daily. Body weight was determined on GD0, 6 and then daily until GD20. Food and water consumption was recorded at designated intervals during pregnancy. On day 20 of pregnancy, females were killed. The terminal body weights of the dams were recorded. The gravid uterus was weighed and foetuses removed by hysterectomy. Females were examined macroscopically. Litter parameters were recorded: number of corpora lutea, implantation sites, early and late resorptions, and dead and live foetuses. Foetuses were weighed, sexed, and submitted to external examination. About half of them were examined for skeletal malformations and half for organ malformations.

Rooting in the bedding material was seen in all rats in the high dose group. Two animals in the high dose group died on GD 20, one day after the last administration of the test material. These deaths were regarded to be treatment related. Eighteen to 24 rats out of 25 per group proved to be pregnant. The body weight development, food and water consumption were not affected by treatment. The terminal body weights were similar in all groups. The uterus weights in the high dose group were slightly, but not significantly lower than in the control or the other groups. This finding was regarded to be accidental. The numbers of corpora lutea, implantations and live foetuses were not affected. The numbers of dead foetuses, and complete, early and late resorptions were not increased. The sex distribution was not affected in any of the groups. The weights of the foetuses in the low, mid, and high dose groups were similar to the control and not affected by treatment. The frequency of all malformations was in a normal range and not increased. The NOAELs for maternal and embryo-foetal toxicity were 15 and 75 mg/kg bw/d, respectively. No teratogenic effects were observed.

 

The developmental toxicity of sodium thioglycolate by the dermal route was evaluated in pregnant rats and rabbits in studies performed according to a method comparable to the OECD guideline 414 (Tyl, 2001a,b; Tylet al., 2003).

Pregnant Sprague-Dawley rats or New Zealand White Rabbits were exposed topically, 6 hr/day, to sodium thioglycolate (99% pure) in vehicle (95% ethanol:distilled water, 1:1) from gestational day (GD) 6 through 19 at dose levels of 0, 50, 100, and 200 mg/kg bw/d for rats orfrom GD 6–29 at 0, 10, 15, 25 or 65 mg/kg bw/d for rabbits.

Twenty-five timed-mated female rats or twenty-four naturally mated female rabbits (in two replicates of 12) were assigned to each group and monitored at regular intervals throughout gestation for clinical signs (including dosing site condition), feed and water intake, and body weight. At necropsy on gestational day 20 or 30, respectively; the following were recorded: maternal clinical condition; body, liver, kidney (rabbits only) and gravid uterine weights; and pregnancy status. The number of ovarian corpora lutea and uterine implantations (resorbed, dead, or live foetuses) was recorded. All live foetuses were counted, weighed, and examined for external alterations, including cleft palate. Approximately 50% of the live rat foetuses or 100% of the live rabbit foetuses per litter were sexed internally and examined for visceral alterations. These foetuses were decapitated and the heads fixed, decalcified, and examined for soft tissue craniofacial alterations. All foetal carcasses were eviscerated (and rat foetuses not scheduled for a visceral examination were sexed internally), macerated, and stained with alizarin red S and alcian blue. Intact rat foetuses (those not decapitated) and all rabbit foetuses were examined for ossified and cartilaginous skeletal alterations.

In rats, sodium thioglycolate treatment was associated with one maternal death at 200 mg/kg/d (only clinical observations at the dosing site preceded death). Other effects, dependent on dose and exposure duration, included body weight and weight gain reductions, changes in relative feed and water intake, and discoloration and slight erythema at the application site. Feed consumption was significantly increased above the control at 50 and 100 mg/kg bw/d, but not at 200 mg/kg bw/d. Maternal water consumption was significantly increased at 200 mg/kg bw/d and slightly (but not statistically significantly) increased at 50 and 100 mg/kg bw/d. Maternal body weights and body weight changes were decreased only at 200 mg/kg bw/d.

At scheduled necropsy, there were no macroscopic indications of organ toxicity. In addition, organ weights and gravid uterine weights were equivalent across groups; maternal body weight was significantly reduced at 200 mg/kg/d. Prenatal viability was unaffected by maternal exposure to sodium thioglycolate. The incidences of foetal external, visceral, and skeletal alterations were also unaffected. A significant, dose-related, upward trend was present for % foetuses with skeletal variations per litter, but there were no significant pairwise comparisons to the vehicle control group value and the % were in the range of the historical control data. Body weights of male and female foetuses per litter at 200 mg/kg bw/d were significantly lower than control values.

The maternal NOAEL could not be identified (<50 mg/kg bw/d), while the developmental toxicity NOAEL was 100 mg/kg bw/d.

In rabbits, no maternal deaths were associated with sodium thioglycolate treatment. One female in the 25 mg/kg bw/d group delivered early and one doe in the 65 mg/kg bw/d group was removed due to a preexisting condition (trichobezoar or hairball in the stomach). Clinical observations were almost exclusively limited to effects of treatmentat the dosing site (skin erythema) in all groups.

Maternal body weight and body weight gain were equivalent across dose groups for all intervals measured except for body weight gain for gestational days 12-15. There was a trend for decreased body weight gain for this interval, which was significant in the high dose group when compared to controls. There were no consistent treatment related reductions in feed consumption.At scheduled necropsy, there was no effect of treatment on terminal maternal body weight. In addition, organ weights and gravid uterine weights were equivalent across groups.

Prenatal viability was unaffected by maternal exposure to sodium thioglycolate. The incidences of foetal external, visceral, and skeletal alterations were also unaffected. Body weights of male and female foetuses per litter and percent males and females per litter were equivalent across dose groups.

The maternal NOAEL for systemic toxicity was at or above 65 mg/kg bw/d; for local toxicity at the dosing site, it was below 10 mg/kg bw/d. The NOAEL for developmental toxicity was >65 mg/kg bw/d.

Toxicity to reproduction: other studies

Additional information

At the end of a sub-chronic (90-day) toxicity studies of sodium thioglycolate administered by cutaneous application to F344/N rats and B6C3F1 mice, a special sperm morphology and vaginal cytology evaluation (SMVCE) was performed (Wolfe and Seung, 2003a,b).

Sperm density and motility, caudal epididymal sperm, spermatid head counts in the testes and testis weights, as well as of vaginal cytology, to allow examination of oestrous cycles, were examined in rats and mice for the controls and three top dose groups.

No differences were observed between the control and treated groups in the female oestrous cycles, in length of cycle, stages, number of females cycling and number of females with regular cycles in either rats or mice. No differences were observed between controls and treated males in sperm parameters or in testicular spermatid counts, or testes and epididymal weights in either rats or mice. However, in mice, the number of sperm per mg cauda, and per total cauda were decreased at 180 and 360 mg/kg bw/d but the reduction was not statistically significant. As the histopathological examination of the testes and epididymis (of rats and mice) performed in these 90-day studies did not show any evidence of damage, this non-significant difference in sperm numbers observed in mice can be dismissed as an incidental finding.

Justification for classification or non-classification

Overall, mercaptoacetic acid and its salts are not considered to be developmental or reproductive toxicants, except at dose levels associated with maternal lethality.

Maternal mortality was observed in the OECD 421 and OECD 416 studies in rats at doses of 40 and 80 mg/kg bw/day. The acute oral LD50 of sodium mercaptoacetate (NaTG) is 50 – 200 mg/kg bw in fasted animals and the lethal doses in the reproductive toxicity studies fall into this range.

Thus, the mortalities of pregnant dams cannot be regarded as reproductive toxicity, but rather a direct consequence of the established toxic mode of action of mercaptoacetates. Mercaptoacetates are known inhibitors of mitochondrial fatty acid β-oxidation. Studies with NaTG have shown that hypoglycaemia occurs in rats dosed with NaTG via oral gavage and that this effect is pronounced in fasted compared to non-fasted animals (Grosdidier 2011, Report No. 37043, IUCLID Section 7.9.4). The hypoglycaemia is a result of impaired gluconeogenesis secondary to inhibition of fatty acid catabolism by mercaptoacetate. Ad-libitum feeding, as employed in the OECD 421 and OECD 416 studies, apparently saves the animals from succumbing already after a single gavage dose by providing a sufficient external glucose supply. Maternal death in NaTG treated dams occurred in late pregnancy (GD 21-23) or early after parturition (PND 1-2). It is likely that the enhanced energy demand due to parturition and / or lactation further exhausts the glucose reserve of the dams ultimately contributing to their death.

Further Information addressing this endpoint and providing additional justification for classification can be found in Section 13.2 Other Assessment Reports (Sullivan Reports, 2006, 2010 and CFCS-Consult, 2017).

A classification as toxic to reproduction is not justified since all maternal and foetal/offspring effects are directly related to the observations leading to a classification as Acute Tox 3 – H301: Toxic if swallowed.