Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target and source substances have similar toxicological properties because:
- all substances are small organic molecules;
- they share structural similarities with common functional groups: one or more thiol and/or thioether group(s) and carboxylic acid (as free acid, salt or ester);
- the metabolism (i.e. ester hydrolysis) leads to comparable products (sulfur-containing core structure in its acid form and alcohols of differing chains lengths)
- covalent protein binding via S-S bonds may be a common mode of action and or chelation of divalent cations via sulfur

The substances were assigned to subgroups according to their main structural features (see Table 1); further justification for subgrouping based on toxicological properties is given below:
- TGA family: Thioglycolic acid, its salts and esters
- 3-MPA family: 3-Mercaptopropionic acid, its salts and esters
- TLA family: Thiolactic acid and its salts
- Intramolecular-S family: Thiodiglycolic acid or Dithiodiglycolic acid and its esters, Thiodipropionic acid or Dithiodipropionic acid and its esters, Methylene bis(butyl thioglycolate)
- Mercaptanes: Thioglycerol, Bis(2-mercaptoethyl) sulfide, 4-Mercaptomethyl-3,6-dithia-1,8-octanedithiol

The acids and salts will dissociate to the respective Thioglycolate or 3-Mercaptopropionate or Thiolactate and the corresponding cation.
In case of the esters, the metabolism expected to occur is ester hydrolysis resulting in the corresponding acid and alcohol.

It was demonstrated, that PETMP and 3-MPA strongly bind to plasma proteins (e.g. via S-S bond to cysteine) in vitro, which is well known for substances containing free SH-groups (Bruno Bock, 2014). Strong protein binding is also expected to occur with the other substances assessed within this paper. The members of the intramolecular-S family are an exception, as they do not contain free SH-groups – protein binding may be less relevant for this family.

Overall, based on close structural similarities, a read-across from the existing repeated dose and reproduction toxicity studies is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see cross-reference -> supporting information

3. ANALOGUE APPROACH JUSTIFICATION
see cross-reference -> supporting information

4. DATA MATRIX
see cross-reference -> supporting information
Cross-referenceopen allclose all
Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across source
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
- 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.
- Food and water: ad libitum
- Acclimation period: 7 days
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.
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.
Oestrous 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
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
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.

Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
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
body weight and weight gain
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
>= 80 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: no effects observed
Dose descriptor:
NOEL
Remarks:
reproductive performance
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
reproductive performance
Dose descriptor:
LOAEL
Remarks:
reprodutive performance
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
reproductive performance
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
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.
Dose descriptor:
NOEL
Remarks:
toxic effects on progeny
Generation:
F1
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
viability
Dose descriptor:
LOAEL
Remarks:
toxic effects on progeny
Generation:
F1
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
viability
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.

Reason / purpose:
read-across source
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
02 February 2009 - 13 October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
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
Remarks:
Doses / Concentrations:
10, 20 and 40 mg a.i./kg/day
Basis:
nominal conc.
dose-level is expressed in active ingredient
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
Oestrous 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
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
F0 generation:
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.

F1 generation:
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)
F0 generation:
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.

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)
F0 generation:
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.

F1 generation:
There were no effects of treatment with the test item on estrous cycles.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
F0 and F1 generations:
There were no effects of treatment with the test item on sperm parameters.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
F0 generation:
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.

F1 generation:
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.

LABORATORY INVESTIGATIONS (F0 generation)
- 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 F0 and F1 parents.

GROSS PATHOLOGY (PARENTAL ANIMALS)
F0 generation:
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.

F1 generation:
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)
F0 generation:
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.

F1 generation:
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.
Dose descriptor:
NOEL
Remarks:
parental toxicity
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
mortality
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Remarks:
parental toxicity
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
mortality
histopathology: non-neoplastic
Dose descriptor:
NOEL
Remarks:
fertility and gestation
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
mortality
Remarks on result:
other: Generation: P and F1
Dose descriptor:
LOAEL
Remarks:
fertility and gestation
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
mortality
Remarks on result:
other: Generation: P and F1
Dose descriptor:
NOEL
Remarks:
fertility
Effect level:
>= 40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
mortality
Remarks on result:
other: Generation: F1 and F2
Dose descriptor:
LOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: Generation: F1 and F2
Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
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
VIABILITY (OFFSPRING)
F1 generation:
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.

F2 generation:
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)
F1 generation:
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.

F2 generation:
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)
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.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
viability
Dose descriptor:
NOEL
Generation:
F2
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
viability
Reproductive effects observed:
no
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.

 

Reason / purpose:
read-across source
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
11-July-2006 - 30-Nov-2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Remarks:
Dept. of Health, UK
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: 8 weeks
- Weight at study initiation: Males: 280 - 379 g; Females: 192 - 260 g
- Housing: All animals were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. During the mating phase, animals were transferred to similar cages on a one male: one female basis.
Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes (Datesand Ltd. Cheshire, UK).
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 28 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55 ± 15 % relative humidity
- Air changes (per hr): 15 x / h
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- The test material was administered by gavage to three groups each of ten male and ten female rats, for up to fifty-four consecutive days, at dose levels of 15, 50 and 150 mg/kg/day.
- A control group of ten males and ten females was dosed with vehicle alone (Deoxygenated and demineralised water).
- Two recovery groups, each of five males and five females, were treated with the high dose (150 mg/kg/day) or the vehicle alone for forty-three consecutive days and then maintained without treatment for a further fourteen days.
Details on mating procedure:
- On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days and smearing commenced.
- Following evidence of mating using vaginal smearing, the males were returned to their original cages and females were transferred to individual cages.
- On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
- Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum.
- Evaluation of each litter size, litter weight, mean pup weight, clinical observations and landmark developmental signs were also performed during this period.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Groups of ten males and females were treated daily. On Day 15 they were paired for a maximum of 14 days, pregnant females were allowed to give birth and maintain their offsprings until Day 5 post partum.
Frequency of treatment:
Each day
Remarks:
Doses / Concentrations:

Basis:
nominal conc.
15, 50 and 150 mg/kg bw/day by gavage
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
none
Parental animals: Observations and examinations:
- Clinical Observations: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before and after dosing, and one hour after dosing at weekends (except for females during parturition where applicable). During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends). All observations were recorded.

- Functional Observations: Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females per non-recovery dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

- Behavioural Assessments: Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation

- Functional Performance Tests: Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions.

- Sensory Reactivity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Startle reflex, Tail pinch, Blink reflex, Finger approach

- Bodyweight: Individual bodyweights were recorded on Day l (prior to dosing) and then weekly for males until termination. Females were weighed weekly until mating was evident. Bodyweights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 postpartum. Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination.

- Food Consumption: During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

- Water Consumption: Water intake was observed daily by visual inspection of water bottles for any overt change.

- Laboratory Investigations: Haematological and blood chemical investigations were performed on five males and five females selected from each non-recovery test and control group on Day 14 (day prior to pairing). Blood samples were obtained from the lateral tail vein. Animals were not fasted prior to sampling.

- Urinalytical investigations were performed on five males selected from non-recovery test and control group during the final week of treatment and on five males selected from each recovery group during the final week of the fourteen day treatment-free period.

- Haematology: The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Het), Erythrocyte indices, Total leucocyte count (WBC), Differential leucocyte count, Platelet count (PLT), Reticulocyte count

- Blood Chemistry: The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Calcium (Ca++), Glucose Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili)

- Urinalysis: The following parameters were recorded on collected urine: Volume, Ketones, Specific Gravity, Bilirubin, pH, Urobilinogen, Protein, Reducing Substances, Glucose, Blood.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
- Testis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of all pups/litter

PARAMETERS EXAMINED
The following parameters were examined in offspring:
- Number of offspring born
- Number and sex of offspring alive recorded daily and reported on Day 1 and 4 postpartum
- Clinical condition of offspring from birth to Day 5 postpartum
- Individual offspring and litter weights on Day 1 and 4 postpartum
- All live offspring were assessed for reflexological response to stimuli by assessing surface righting reflex on Day 1 post partum.


GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

ORGAN WEIGHTS:
- These reproductive organs were weighed from all animals that were killed at the end of the study: Ovaries, testes, epididymides
- The following organs, removed from five selected males and parental females from each group that were killed at the end of the study, were dissected free from fat and weighed before fixation: Adrenals, liver, brain, ovaries, epididymides, spleen, heart, testes, kidneys, thymus

HISTOPATHOLOGY:
- From all animals: Ovaries, pituitary, prostate, coagulating gland, seminal vesicles, epididymides, testes, uterus/cervix
- From five selected males and females of each group that were killed at the end of the study: Adrenals, ovaries, aorta (thoracic), pancreas,bone & bone marrow (femur including stifle joint), pituitary, bone & bone marrow (stemum), prostate, brain (including cerebrum, cerebellum and pons), oesophagis, caecturi, rectum, coagulating gland, salivary glands (submaxillary), colon, sciatic nerve, duodenum, seminal vesicles, epididymides, skin (hind limb), eyes, spinal cord (cervical), gross lesions, spleen, heart, stomach, ileum, thyroid/parathyroid Jejunum, trachea, kidneys, testes, liver, thymus, lungs (with bronchi), urinary bladder, lymph nodes (cervical and mesenteric), uterus/cervix, mammary gland, vagina, muscle (skeletal)
Postmortem examinations (offspring):
Not examined
Statistics:
- Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight),
weekly bodyweight gain, litter weights, offspring bodyweights and quantitative functional
performance data were assessed for dose response relationships by linear regression analysis,
followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity
of variance.
- Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test.
- Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.
- The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and
landmark developmental markers.
- The haematology variable basophils was not analysed since consistently greater than 30% of the
data were recorded as the same value.
Reproductive indices:
Pre-coital interval and fertility indices were calculated
Offspring viability indices:
Live birth and viability indices were calculated
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
MORTALITY: There were five interim deaths during the study, these were confined to females treated with 150 mg/kg/day. One female (number 76) died during the maturation phase on Day 10. A second female (number 73) was killed in extremis on Day 23 (Day 7 of gestation). Two females were found dead on their Day 22 of gestation (Day 40 of the main study for female 80 and Day 41 of the main study for female 75), The final of the five females (number 72) was killed in extremis on it’s Day 1 of lactation phase (Day 41 of main study).

CLINICAL SIGNS: The only toxicological significant observations were detected for the two interim death females that were killed in extremis (numbers 73 and 72), these included incidents of tip-toe gait, hunched posture and pilo-erection. One of the females (number 73) also had red/brown staining around the mouth. The deterioration of the females’ condition subsequently led to their termination. No toxicologically significant findings were detected for animals of either sex after the treatment period, or for animals of either sex after the fourteen-day recovery period.

BODY WEIGHT AND WEIGHT GAIN: Females treated with 150 mg/kg/day showed a reduction in group mean bodyweight and bodyweight gain between Day 0 and Day 7 of gestation.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Food consumption was reduced for females treated with 150 mg/kg/day
during Days 0 to 7 of gestation when compared to controls although statistical significance was not achieved.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No overt intergroup differences were detected for all animals after the
treatment period


HAEMATOLOGY: No treatment-related effects were detected for the haematological parameters investigated


CLINICAL CHEMISTRY: No treatment-related intergroup differences in blood chemical parameters were detected


URINALYSIS: No toxicologically significant effects were detected in treated animals when compared to controls


NEUROBEHAVIOUR: No treatment-related effects were detected for animals of either sex from all treatment groups after the treatment period. Formal behavioural assessments were not carried out for recovery group animals.


ORGAN WEIGHTS: No treatment-related intergroup differences in organ weights were detected for all treated animals after the treatment period


HISTOPATHOLOGY: There were no treatment-related microscopic changes detected for the pituitary or reproductive tissues examined for terminal kill animals after the treatment period.here were five unscheduled deaths during the course of the study. Three females were found dead and two were killed in extremis, all five females were from the 150 mg/kg/day treatment group. With the possible exception of pyometria reported for femal number 72 and focal gastric ulceration for female 73, factors contributing to the death or moribundity in these animals were not evident from histopathological evaluation




Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no effects observed
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: clinical signs; mortality; body weight
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
- No intergroup differences were observed

CLINICAL SIGNS (OFFSPRING)
- There were no clinically observable signs of test material toxicity detected in offspring from treated animals.

BODY WEIGHT (OFFSPRING)
- Offspring weight at Day 1 of lactation was reduced in the 150 mg/kg/day dose group. This was due to a single dam (number 72) and litter that was killed in extremis on Day 1 of lactation due to the deterioration of the physical health of the dam. The poor condition of the dam led to the reduced bodyweight detected in the animal’s offspring, thus, leading to the reduction in group mean bodyweight for offspring on Day 1 lactation. The offspring bodyweight for the remaining 150 mg/kg/day litters were comparable to controls at Day 4 of lactation. The actual bodyweight and the offspring bodyweight change between Day 1 and 4 of lactation for the remaining litters in the 150 mg/kg/day treatment group were comparable to controls, therefore, this slight difference in offspring bodyweight was considered to be a result of the poor condition of a single dam in the 150 mg/kg/day treatment group and not a representation of reproductive toxicity.

SEXUAL MATURATION (OFFSPRING)
- No treatment-related effects on offspring development were detected.

OTHER FINDINGS (OFFSPRING)
- No intergroup differences were observed for litter size or sex ratio
- No treatment related macroscopic abnormalities were detected for the interim death offspring or for the remaining offspring at terminal kill.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects on fertility or on testicular weight and histopathology
Reproductive effects observed:
no
Conclusions:
The oral administration of GMT 80% to rats by gavage, at dose levels of 15, 50 and 150 mg/kg bw/d, resulted in no treatment related effects for the males treated with 150 mg/kg bw/d but did result in the death of five females treated with 150 mg/kg bw/d. The NOAEL for systemic toxicity was considered to be 150 mg/kg bw/d for males and 50 mg/kg bw/d for females. The NOAEL for reproductive effects in the males is also 150 mg/kg bw/d as assessed by the absence of adverse effects on fertility or on testicular weight and histopathology. Due to the high matemal mortality and toxicity at 150 mg/kg bw/d in the females, which precludes reproductive assessment at that dose level, the NOAEL for reproductive toxicity in females is 50 mg/kg bw/d.
Reason / purpose:
read-across source
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 9 february to 6 July 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, North Carolina, USA
- Age at initiation of dose administration: approximately 10 weeks old
- Weight at initiation of dose administration: Males: 322.9-387.3 g; Females: 210.3-261.5 g;
- Housing: individually
- Diet: Certified Rodent Labdiet 5002 ad libitum
- Water: Reverse osmosis-purified (on-site) drinking water ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5-21.6
- Humidity (%): 35.7-40.7
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 9 february 2005 To: 6 July 2005
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test article formulations were weight/volume (test article/vehicle) formulations. They were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation and stored at room temperature. They were stirred continuously throughout the
preparation, sampling and dose administration procedures. They were visually found to be homogeneous and anatically confirmed to be homogeneous.


VEHICLE
- Justification for use and choice of vehicle (if other than water): no
- Concentration in vehicle: 0 (vehicle group), 2 (low), 10 (middle) and 30 mg/mL (high concentration group)
- Amount of vehicle (if gavage): Dosage volume was 5 mL /kg for all groups
- Purity: 100%
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear, referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): in plastic maternity cages with nesting material, ground corncob bedding.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Resuspension homogeneity and 11-day stability were established in a previous study. Samples for concentration analysis were collected from the
middle stratum of each formulation (including control) every 2 weeks.
Duration of treatment / exposure:
Exposure period: Males received 15 daily doses prior to mating. Males were dosed throughout the mating period for a total of 54 doses.
Females received a minimum of 15 daily doses prior to pairing and were dosed through lactation.
Duration of test substance (TS) exposure: males 54 days; females 41-44 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
10, 50 and 150 mg/kg bw /day
Basis:
nominal conc.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected based on the results of a previous study (See Bowman (2005)/Oral 14 day repeated 02). In that study, 1 female in the 150 mg/kg/d group was found dead on study day 2. Effects on the mean bodyweight, on food consumption and liver and kidney weights were also observed at this level in both sexes. Increased liver and kidney weights were also observed in the males of the 50 mg/kg/d group.
- Rationale for animal assignment: no (randomization).
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data. Cage-board was changed 3 times per week.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly and twice daily for mortality or signs of moribundity


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly until pairing; not recorded during mating period; then weekly for males and
on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4.

WATER CONSUMPTION : No
Oestrous cyclicity (parental animals):
During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. Individual gestation length was calculated using the date delivery started.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
testis weight, epididymis weight
Litter observations:
Pups were examined for gross malformations, and the number of stillborns and alive pups were recorded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at day 54 after the first day of treatment
- Maternal animals: All surviving animals at post-mating day 25 or at lactation day 4


GROSS NECROPSY
- Gross necropsy consisted of examination of external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic,
abdominal and pelvic cavities, including viscera.


HISTOPATHOLOGY / ORGAN WEIGHTS
Ovaries with oviducts, pituitary gland, testes, epididymides were prepared for microscopic examination and weighed. Brain, kidneys and liver were weighed. Coagulating glands, mammary gland, prostate gland, seminal vesicle, uterus with vagina and cervix and all gross lesions were prepared for microscopic examination.
Selected tissues were examined microscopically from all F0 animals in the control and high-dose groups and females in the low- and mid-dose
groups.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age following an external examination and discarded without further evaluation.
Statistics:
Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test
group to the control group by sex. Statistical analyses were not conducted if the number of animals was two or less. Data obtained from nongravid
females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.
Parental mating, fertility, conception and copulation indices were analyzed using the Chi-square test with Yates' correction factor
(Hollander and Wolfe, 1999). Mean parental body weights (weekly, gestation and lactation), body weight changes and food consumption,
offspring body weights and body weight changes, gestation length, number of implantation sites, number of corpora lutea, number of pups born,
live litter size on Post Natal Day (PND) 0, unaccounted-for sites, absolute and relative organ weights, and pre-coital intervals values were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran. 1980) to determine intergroup differences. If the ANOVA revealed
statisticallv significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA test
(Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance,
Dunn's test (Dunn, 1964) was used to compare the test groups to the control group.
Reproductive indices:
Mating, fertility and copulation/conception indices were calculated.
Offspring viability indices:
Mean live litter size and postnatal survival were calculated.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Three males in the 150 mg/kg/day group were found dead or euthanized in extremis due to excessive body weight loss on study days 4 or 14. Two of them had decreased defecation and an unkempt appearance the day of or prior to euthanasia on study day 14.
Three females in the 150 mg/kg/day group were found dead or euthanized in extremis on gestation day 21 or 22. On gestation day 22, one female
exhibited signs of dystocia: hypoactivity, an unkempt appearance, piloerection, soft stool, drooping eyelids, hypothermia (cool to the touch) and was recumbent and unresponsive to handling; consequently, this female was euthanized later that day.

Salivation and/or clear material around the nose and/or mouth was observed in the 50 mg/kg/day group males and the 150 mg/kg/day group
males and females at the time of dose administration and/or approximately 1-2 hours following dose administration.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males:
Mean body weight gain was reduced (statistically significant, p<0.01) in the 150 mg/kg/day group during the pre-mating period (study days 0-14) compared to that in the control group, resulting in reduced (not statistically significant) mean body weight gains when the entire treatment period
(study days 0-54) was evaluated. An increase in food consumption was observed during the post-mating period. It was attributed to the weight loss.
Mean body weights and body weight gains in the 10 and 50 mg/kg/day group males were similar to those in the control group throughout the study. None of the differences were statistically significant.

Females:
No statistical change was elicited during pre-mating and lactation periods.
During gestation days 17-20, mean body weight gains in the 150 mg/kg/day group females were reduced compared to those in the control group.
The difference was statistically significant (p<0.05).
No statitical difference in food consumption was attributed to treatment.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Mean gestation lengths in the 10, 50 and 150 mg/kg/day groups were similar to those in the control group. No statistically significant differences
were noted.
In one female at 150 mg/kg bw/d, signs of dystocia were observed. In addition, 2 other females in this group were found dead l day prior to the expected day of parturition (gestation day 21).

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No significant effect of treatment on testes and epididymides weight was observed.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test article-related effects on F0 reproductive performance were observed at any dosage level.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Test article-related increases in mean relative (to final body and brain weight) liver weights were observed in the 150 mg/kg/day group males and
females. The difference from the control group relative (to final body weight) value was statistically significant (p<0.01) in the males and females. The increase in mean relative liver weight corresponded to the microscopic findings observed in the 150 mg/kg/day group animals that were found
dead or euthanized in extremis. An increase in mean kidney weight (absolute and relative to final body and brain weights) was observed in the 150 mg/kg/day group males; the difference from the control group relative (to final body weight) value was statistically significant (p<0.01).

GROSS PATHOLOGY (PARENTAL ANIMALS)
Three males and 3 females in the 150 mg/kg/day group were found dead or euthanized in extremis during the study.
One male was found dead on study day 4 and had no gross pathology findings. The two other males were euthanized in extremis on study day 14; both males had pale livers. One also had white areas in the liver.
One dead female had a white area in the liver and dark red discoloration of the lungs; there were no other test article-related macroscopic findings.
The findings in the liver corresponded to increases in the mean relative liver weights in males and females in the 150 mg/kg/day group at the
scheduled necropsy, and were therefore considered test article-related.
No other significant findings were observed by macroscopic examination.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The 2 males that were euthanized in extremis and 1 of the females that was found dead had test article-related findings (periportal or generalized
hepatocellular vacuolation) in the liver. This finding was characterized by the presence of numerous small, round, clear vacuoles within the
hepatocellular cytoplasm, which distended the affected cells and gave the cytoplasm a foamy appearance. Affected cells were limited to the areas
immediately surrounding portal triads (periportal hepatocellular vacuolation) or affected hepatocytes in a diffuse manner without apparent zonal
distribution (generalized hepatocellular vacuolation). This finding correlated with the macroscopic findings (white areas or pale liver) observed
in these animals that were found dead/euthanized in extremis and with increased relative liver weights observed in animals at the scheduled
necropsy.
Livers were not scheduled to be retained/examined at the scheduled necropsy. Therefore, further investigation of this finding at the
scheduled necropsy was not possible.

Mucification of the cervical and vaginal epithelium was noted microscopically in the 150 mg/kg/day group gravid females that died or were euthanized in extremis prior to the scheduled necropsy on lactation day 4. This finding may be a morphologically normal peri-parturitional phenomenon, but since there were no control group animals examined at that age, this cannot be unequivocally established. However. there was a dose-related increase in incidence and severity of mucification compared to the control group at the scheduled necropsy on lactation day 4 (1/11 (9.1%),
3/12 (25%), 3/10 (30%) and 4/6 (67%) of the gravid females in the control, 10, 50 and 150 mg/kg/day groups, respectively). Taken in context of the maternal mortality, dystocia and adverse effects on pup growth and survival, mucification of the vaginal epithelium in the 150 mg/kg/day group was considered test article-related.

All other microscopic changes were consistent with normal background lesions in clinically normal rats of the strain and age used in this study, and were considered to be spontaneous and/or incidental in nature and unrelated to test article administration.


Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: mortality, body weight, food consumption, organ weights, histopathology.
Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
Mean live litter size on Post Natal Day (PND) 0 in the 150 mg/kg/day group (12.7 pups per dam) was decreased compared to the control group value (15.3 pups per dam). The difference was not statistically significant.

When the entire postnatal period (birth to PND 4) was evaluated, postnatal survival in the 150 mg/kg bw/d group (73.2% per litter) was reduced compared to that in the control group (98.9% per litter). The difference was statistically significant (p<0.05).
The effects on postnatal survival in this 150 mg/kg/day group were considered test article-related.

The mean number of pups born in the 150 mg/kg/day group (13.3 per dam) was slightly (not statistically significant) lower than the control group
value (15.5 per dam). This reduction was due to one female that delivered only 8 pups: this female also had only 8 corpora lutea. Therefore, the
reduction in the mean number of pups born (not survival) in the 150 mg/kg/day group was not considered to be treatment-related, although there was clear maternal toxicity (mortality) in this dose group.

The percentage of males at birth in the 150 mg/kg/d group was unaffected by administration of the test article to the parental animals.

The mean number of pups born, live litter size and the percentage of males at birth in the 10 and 50 mg/kg/day groups were similar to the control
group values. Postnatal survival in these groups were unaffected by test article administration.


CLINICAL SIGNS (OFFSPRING)
2(2), 2(2), 2(2) and 23(4) pups (litters) were found dead in the control, 10, 50 and 150 mg/kg/d groups, respectively. In the 150 mg/kg/day group, 4 pups were missing and presumed to have been cannibalized and 7 pups were small; these findings were considered to be test article-related. The general physical condition of pups in the 10 and 50 mg/kg/day groups was unaffected by test article administration.

BODY WEIGHT (OFFSPRING)
Mean body weights in the 150 mg/kg/day group pups were decreased compared to the control group values (10.0% - 17.2% for males and 13.6% - 18.3% for females on PND 1 and 4); the majority of the differences were statistically significant (p<0.05). Mean male and female pup body weight gains
during PND 1-4 in this group were reduced compared to those in the control group: the difference was not statistically significant. The reductions in pup body weights and body weight gains were attributed to the test article.

GROSS PATHOLOGY (OFFSPRING)
Of the pups (litters) found dead during PND 0-4, 1(1), 0(0), 1(1) and 18(2) were too autolyzed to examine in the control, 10, 50 and 150 mg/kg/day groups, respectively.
Of the remaining pups (litters), 1(1), 2(2), 1(1) and 5(2) had no presence of milk in the stomach in the control, 10, 50 and 150 mg/kg/day groups, respectively.
No other gross pathology findings were noted.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: viability index
Reproductive effects observed:
not specified

Table 1 Mean Body Weights (g) – Parental Males

Exposure Group

Control

10 mg/kg/d

50 mg/kg/d

150 mg/kg/d

Males

Initial

357.1 +/- 16.6

356.0 +/- 13.8

355.5 +/- 14.6

353.7 +/- 13.9

Week 1

389.9 +/- 23.7

384.3 +/- 20.4

383.1 +/- 16.6

369.4 +/- 25.3

Week 2

419.6 +/- 29.6

411.3 +/- 24.6

411.0 +/- 16.3

367.1 +/- 54.9**

Week 3

436.4 +/- 16.6

427.8 +/- 28.7

427.0 +/- 17.8

405.8 +/- 21.9

Week 4

463.6 +/- 33.4

453.7 +/- 30.9

451.3 +/- 18.5

427.0 +/- 21.7*

Week 5

489.1 +/- 39.0

477.4 +/- 32.9

470.2 +/- 26.8

449.1 +/- 27.9

*Statistically significant difference from the control group (p<0.05) using Dunnett’s test.

**Statistically significant difference from the control group (p<0.01) using Dunnett’s test.

Table 2 Mean Body Weights changes (g) – Statistical changes in Parental animals

Exposure Group

Control

10 mg/kg/d

50 mg/kg/d

150 mg/kg/d

Males

Day 0-7

32.9 +/- 10.2

28.3 +/- 8.4

27.6 +/- 7.9

17.0 +/- 17.7**

Day 0-14

62.3 +/- 15.8

55.3 +/- 13.6

55.6 +/- 10.5

14.8 +/- 51.8**

Females

Day 0-7

9.1 +/- 5.2

8.9 +/- 7.6

9.6 +/- 7.1

0.4 +/- 11.7*

Day 7-14

8.4 +/- 8.2

9.2 +/- 5.7

9.2 +/- 5.9

26.8 +/- 19.8**

Gestation Day 17-20

44 +/- 6.6

43 +/- 8.4

47 +/- 7.3

30 +/- 21.8*

*Statistically significant difference from the control group (p<0.05) using Dunnett’s test.

**Statistically significant difference from the control group (p<0.01) using Dunnett’s test.

Table 3 Relative Organ Weights – Statistical changes in Parental animals scheduled for necropsy

Exposure Group

Control

10 mg/kg/d

50 mg/kg/d

150 mg/kg/d

Organ Weight (g) to bodyweight

Males

Kidney

0.714 +/- 0.048

0.694 +/- 0.043

0.739 +/- 0.070

0.795 +/- 0.067**

Liver

3.77 +/- 0.32

3.72 +/- 0.31

3.98 +/- 0.38

4.33 +/- 0.34**

Females

Kidney

0.695 +/- 0.046

0.700 +/- 0.058

0.698 +/- 0.043

0.763 +/- 0.046

Liver

4.48 +/- 0.42

4.41 +/- 0.35

4.45 +/- 0.34

5.20 +/- 0.42**

**Statistically significant difference from control group (p<0.01) using Dunnett’s test.

Table 4 Postnatal survival rates (Birth to Postnatal Day 4)

Exposure Group

Control

10 mg/kg/d

50 mg/kg/d

150 mg/kg/d

% per litter

Birth to Postnatal Day 4

98.9 +/- 2.5

97.9 +/- 3.9

98.7 +/- 2.7

73.2 +/- 33.9

**Statistically significant difference from control group (p<0.01) using Dunnett’s test.

Table 5 Mean Body Weights (g) – Offspring

Exposure Group

Control

10 mg/kg/d

50 mg/kg/d

150 mg/kg/d

Males

Postnatal Day 1

7.0 +/- 0.6

6.8 +/- 0.4

7.1 +/- 0.8

6.3 +/- 0.7

Postnatal Day 4

9.9 +/- 1.1

9.2 +/- 0.7

9.6 +/- 1.4

8.2 +/- 0.7*

Females

Postnatal Day 1

6.6 +/- 0.8

6.4 +/- 0.3

6.7 +/- 0.7

5.7 +/- 0.7*

Postnatal Day 4

9.3 +/- 1.2

8.7 +/- 0.7

9.2 +/- 1.1

7.6 +/- 1.3**

*Statistically significant difference from control group (p<0.05) using Dunnett’s test.

Conclusions:
A dosage level of 50 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity resulting from exposure to 2-ethylhexyl mercaptoactetate when administered orally by gavage to rats.
Executive summary:

This study was designed to provide information on the potential adverse effects of oral exposure 2-ethylhexyl mercaptoacetate on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behavior, conception, parturition and lactation of the Fo generation and development of offspring from conception through day 4 of postnatal life.

2-ethylhexyl mercaptoacetate, in the vehicle, corn oil, was administered orally by gavage once daily to 3 groups of Crl:CD (SD) rats, each group consisting of 12 males and 12 females. Dosage levels were 10, 50 and 150 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 12 rats/sex received the vehicle on a comparable regimen. Males received 15 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 54 doses. Females received a minimum of 15 daily doses prior to pairing and were dosed through lactation day 3 for a total of 41-44 doses.

In this study, severe parental systemic toxicity was observed in the 150 mg/kg/day group. It was characterized by: mortality, moribundity, decreased mean body weight gain, decreased consumption of feed, increased liver and kidney weight, or hepatocellular vacuolization in at least one sex of the F0 animals; and increased mucification of the cervical and vaginal epithelium in post-partum F0 dams. Decreased viability and growth of the F1 animals through post-partum day 4 also occurred at the 150 mg/kg/day dose. Within the limits of the experimental design, a dosage level of 50 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity resulting from exposure to 2-ethylhexyl mercaptoactetate when administered orally by gavage to rats.

Reason / purpose:
read-across source
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
06-JUN-2006 to 13-JUN-2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Source: RCC Ltd, Laboratory Animal Services, Wölferstrasse 4, CH-4414 Füllinsdorf / Switzerland
- Age at study initiation: 10 weeks
- Weight at study initiation: Males: 286 - 331 grams, Females: 180 - 207 grams
- Fasting period before study: only before blood sampling
- Housing: Animals were housed in Makrolon cages (type-3) with wire mesh tops and standard granulated softwood bedding. During the pre-pairing period, males and females were housed individually. Cages of males were interspersed amongst those holding females to promote the development of regular estrus cycles. During the pairing period, rats were housed one male/one female in Makrolon pairing cages. After mating or at the end of the pairing period, the males and the females were housed individually again. During the lactation period (until day 4 of lactation), dams were housed together with their litters.
- Food and water: ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 13-JUN-2006 To: 30-JUL-2006
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer a homogenous mixture was prepared. Having obtained a homogenous mixture, vehicle was added until the required final volume was achieved. Separate formulations were prepared for each concentration.
During the daily administration period homogeneity of the test item in the vehicle was maintained using a magnetic stirrer.
VEHICLE
- Corn oil is the vehicle of choice for substances with low water solubility
- Concentration in vehicle: 6.25, 12.50, 25.00 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
Details on mating procedure:
After the animals had received the test item for 14 days, the pairing period was initiated while dosing was continued.
During the pairing period females were housed with males (one male : one female) in special automatic mating cages, i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed.
This system reduces the variation in the copulation times of the different females.
Females were removed and housed individually if:
a) a copulation plug was observed, and / or
b) the daily vaginal smear was sperm-positive.
This day was designated day 0 post coitum.
Females showing no-evidence of copulation were sacrificed 24-26 days after the last day of the pairing period and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for determination of concentration, homogeneity and stability (7 days) of the dose formulations were taken during the first week of the administration period. Additionally, samples for determination of concentration and homogeneity were taken during the last week of the administration period.
On each occasion three samples of approximately 2 g were taken from the top, middle and bottom of each formulation and transferred into flat bottomed flasks. The samples were frozen (-25°C to -15°C) pending analysis. Samples were sent on dry ice to Dr. D. Flade, RCC Ltd, Environmental Chemistry & Pharmanalytics, CH-4452 Itingen / Switzerland. Analysis was performed using a method developed by RCC Ltd. After analysis, the analytical results were communicated to the Study Director. Upon receipt and evaluation of these results the Study Director decided about discarding the samples.
Duration of treatment / exposure:
Exposure period: at least 28 days (males); for 14 days prior to pairing, through pairing and gestation until day 4 post partum (females).
Premating exposure period (males): 14 days
Premating exposure period (females): 14 days
Duration of test: up to 28 days in males; up to 51 days in females
Frequency of treatment:
daily
Details on study schedule:
see Table 1 (Materials & Methods)
Remarks:
Doses / Concentrations:
25, 50 and 100 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: none
- Dose selection rationale: Dose levels were selected in agreement with the Sponsor, based on the results of a dose range-finding study (RCC Study No. A57802) and following discussions with the sponsor.
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality, clinical signs. Additionally, the females were observed for signs of difficult or prolonged parturition.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first test item administration and weekly thereafter.


BODY WEIGHT: Yes
- Time schedule for examinations: daily


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day before or on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: 5 per sex per group
- Parameters:
Erythrocyte count, Hemoglobin concentration distribution width, Haemoglobin Platelet count, Haematocrit Total leukocyte count, Mean corpuscular volume, Differential leukocyte count, Red cell volume distribution width, Mean corpuscular hemoglobin concentration, Mean corpuscular haemoglobin
Coagulation: Thromboplastin time, Activated partial thromboplastin time



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:on the day before or on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 5 per sex per group
- Parameters:
Glucose, Sodium, Urea, Potassium, Creatinine, Chloride, Bilirubin, total Calcium, total Cholesterol, inorganic Phosphorus, Aspartate aminotransferase, total Protein, Alanine aminotransferase, Albumin, Bile acids, Globulin, Alkaline phosphatase, Albumin/Globulin ratio, Gamma-glutamyl-transferase


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At one time during the study (males: shortly before scheduled sacrifice; females: on day 3 or 4 post partum) relevant parameters were evaluated for five P generation males and five P generation females randomly selected from each group.
- Dose groups that were examined: all
- Battery of functions tested: Cage side observations, Hand-held observations, Open field observations, Categorical observations, Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.
Oestrous cyclicity (parental animals):
no
Sperm parameters (parental animals):
no
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
litter size, live birth, stillbirth and any gross anomalies. The sex ratio of the pups was recorded. The dams were caged together with their litters until day 4 of lactation. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.
The dams and pups were observed daily for survival and behavioural abnormalities in nesting and nursing.

GROSS EXAMINATION OF DEAD PUPS:
yes
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
The testes and epididymides of all parental males were weighed.
In addition for five adult males and females, randomly selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken:
liver, spleen, adrenals, brain, thymus, heart, kidneys

HISTOPATHOLOGY: Yes
prostate, testes, seminal vesicles with coagulation gland, epididymides, ovaries, gross lesions, heart, brain, thymus, spinal cord, thyroid, small and large intestines (incl. Peyer's patches), trachea and lungs, stomach, uterus (with vagina), liver, urinary bladder, kidneys, lymph nodes, adrenals, peripheral nerve, spleen, bone marrow
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to macroscopic postmortem examination.


GROSS NECROPSY
- Gross necropsy consisted of external examinations.
Statistics:
The following statistical methods were used to analyze body weights, food consumption, reproduction and skeletal examination data:
• Means and standard deviations of various data were calculated and included in the report.
• If the variables could be assumed to follow a normal distribution, the Dunnett t-test, based on a pooled variance estimate, was used for intergroup comparisons (i.e. single treatment groups against the control group).
• The Steel test (rank test) was applied when the data could not be assumed to follow a normal distribution.
• Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
Mating index: no. of females mated/ no. of females paired
Fertility index: no. of pregnant females / no. of females mated
Gestation index: no. of litters with live pups / no. of pregnant females
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All animals survived until scheduled necropsy. In group 4, all animals pushed their heads through the bedding after administration of the test item starting on day 5 of the prepairing period and continuing until the end of the treatment period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
unaffected

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
no data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
no data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): see Table 2

ORGAN WEIGHTS (PARENTAL ANIMALS): see Table 3
For groups 3 and 4 males, liver weights relative to body weights and brain weights were dose-dependently increased. Liver weights relative to the brain weights did not reach statistical significance in group 3.
In the absence of a histopathological correlation these higher weights were considered to be of no adverse character.
In females, mean absolute organ weights as well as organ/body weight ratios and organ/brain weight ratios were not affected by exposure to the test item.

GROSS PATHOLOGY (PARENTAL ANIMALS)
During necropsy of parent animals no test item-related findings were noted.

HISTOPATHOLOGY (PARENTAL ANIMALS): see Table 4
A minimal to slight hyperplasia of the forestomach squamous epithelium partly associated with a minimal to slight hyperkeratosis and minimal inflammatory cell infiltrations was recorded in four males and four females in group 4. Proliferative lesions of the rodent non-glandular stomach region are relatively common in gavage and feeding studies ranging from mild hyperplasia of the keratinized stratified squamous epithelium to extensive papillomatous hyperplasia. As no similar findings were noted in the forestomach epithelium of the control group, this finding was considered to be test item-related.
All other microscopic findings recorded in various organs of all groups treated with the test item did not differ significantly from the control group. All findings were considered to be spontaneous in nature and within the normal background pathology commonly seen in rats of this strain and age.
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
not measured/tested
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Reproductive effects observed:
no

Result: no effects on reproduction at the highest dose tested (100 mg/kg bw/d)
The fertility rate was high resulting in 9, 8, 9 and 10 litters in order of ascending dose levels for evaluation of reproduction data. At all concentrations, there were no treatment-related effects on precoital time, fertility indices, mean duration of gestation, number of implantations, or post-implantation loss. The mean number of
corpora lutea per dam (determined at necropsy) was similar in all groups and gave no indication of a test item-related
effect.

Table 2: Reproductive performance

 

Group 1

0 mg/kg/d

Group 2

25 mg/kg/d

Group 3

50 mg/kg/d

Group 4

100 mg/kg/d

Mating index (%)

100.0

100.0

100.0

100.0

Fertility index (%)

90.0

80.0

90.0

100.0

Conception rate (%)

90.0

80.0

90.0

100.0

Gestation index (%)

 100.0

100.0

100.0

100.0

 

Table 3: Organ weights P-generation males

MALES

Group 1

0 mg/kg/d

Group 2

25 mg/kg/d

Group 3

50 mg/kg/d

Group 4

100 mg/kg/d

BODY W. [g]

342.3

360.3

352.1

355.9

ST.DEV.

17.8

17.2

23.9

23.4

N

10

10

10

10

BRAIN [g]

2.02

2.01

2.08

1.97

% BW

0.60

0.57

0.59

0.57

N

5

5

5

5

HEART [g]

0.95

1.02

1.04

1.01

% BW

0.28

0.29

0.29

0.29

N

5

5

5

5

LIVER[g]

8.30

8.62

9.40

9.38

% BW

2.46

2.43

2.65*

2.70*

N

5

5

5

5

THYMUS[g]

0.306

0.364

0.287

0.348

% BW

0.091

0.103

0.082

0.099

N

5

5

5

5

KIDNEYS[g]

2.26

2.23

2.34

2.40

% BW

0.67

0.63

0.66

0.69

N

5

5

5

5

ADRENALS[g]

0.074

0.081

0.081

0.088

% BW

0.022

0.023

0.023

0.025

N

5

5

5

5

SPLEEN[g]

0.69

0.72

0.76

0.68

% BW

0.21

0.21

0.22

0.20

N

5

5

5

5

TESTES[g]

3.63

3.69

3.76

3.83

% BW

1.06

1.02

1.07

1.08

N

10

10

10

10

EPIDIDYMIDES[g]

1.225

1.231

1.240

1.275

% BW

0.358

0.342

0.352

0.359

N

10

10

10

10

*/**: Dunnett-test based on pooled variance sig. at 5% or 1% level.

Table 4: Histopathology

Dose group

1

2

3

4

1

2

3

4

Sex

Males

Females

Stomach

No. examined

5

5

5

5

5

5

5

5

epithelial hyperplasia

4

4

grade 1

1

1

grade 2

3

3

hyperkeratosis

3

1

grade 1

3

grade 2

1

mononuclear infiltrate

1

1

2

grade 1

1

1

2

inflammation

2

3

grade 1

2

3

Conclusions:
No treatment-related effects on reproduction were evident.
The NOEL for reproductive toxicity was considered to be 100 mg/kg bw/d.
Reason / purpose:
read-across source
Reference
Endpoint:
fertility, other
Remarks:
extended OECD TG 408 assessing reproductive parameters
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents) (30 September 1996)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents) (21 September 1998)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: RccHan: WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Ca. 7 weeks at delivery
- Weight at study initiation: Males: 162 - 203 g (mean: 181 g) / Females: 125 - 151 g (mean: 138 g)
- Housing: In groups of two to five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding including paper enrichment
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2914C rodent maintenance diet, ad libitum
- Water (e.g. ad libitum): Community tap-water, ad libitum
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%):30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). The mixture was mixed thoroughly using a magnetic stirrer until homogeneous. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
The dose formulations were stored for up to eight days in glass beakers in a refrigerator at 2 - 8 °C.


VEHICLE
- Concentration in vehicle: 0/ 2.5 / 10 / 40 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight
Details on mating procedure:
animals were not mated
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
-sampling: at start, on days 1, 42 and 85 of treatment, duplicate samples of the control group as well as three samples (top, middle and bottom) were taken prior to dosing for analysis of homogeneity and concentration / duplicate samples were taken at experimental start to confirm stability (4 hour and 8 days)
- samples were stored there at -15 - 25 °C until analysis.
- analysis by HPLC coupled to a UV detector (215 nm)
Duration of treatment / exposure:
treatment: 91/92 days; recovery: 28 d
Frequency of treatment:
daily
Details on study schedule:
animals were not mated
Remarks:
Doses / Concentrations:
0, 12.5, 50, 200 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10 (control group), 10 (treatment groups), 5 (recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on dose range finding study
- Post-exposure recovery period in satellite groups: 4 weeks

- Dose selection rationale: based on results obtained in a 14-day dose range-finding study
- Rationale for animal assignment (if not random): Randomly allocated to groups by body weight.
- Post-exposure recovery period in satellite groups: 28 d
Parental animals: Observations and examinations:
Viability / Mortality: Twice daily
Clinical signs: once daily during accimatisation; Twice daily on days 1 to 3; once daily thereafter; once daily during recovery period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly (weeks 1-12)

BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once in week 13 and 17 (recovery groups)
- Dose groups that were examined:
main + recovery groups: animals of the control and high dose groups as well as in animals of the middle dose groups if test item-related changes are seen in the high dose

HAEMATOLOGY: Yes
- Time schedule for collection of blood: main + recovery groups: week 13 / recovery groups: week 17
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Reticulocyte count, Reticulocyte maturity index (low, medium, high fluorescence), total Leukocyte count, Differential leukocyte count (Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes) Large unstained cells, Platelet count, Prothrombin time (= Thromboplastin time), Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: main + recovery groups: week 13 / recovery groups: week 17
- Animals fasted: Yes
- How many animals: all
- Parameters: Glucose, Urea, Creatinine, toral Bilirubin, total Cholesterol, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Alkaline phosphatase, Gamma-glutamyl-transferase, Creatine kinase, Sodium, Potassium, Chloride, Calcium, Phosphorus, total Protein, Albumin, Globulin, Albumin/Globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: main + recovery groups: week 13 / recovery groups: week 17
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters: Urine volume (18 hour), Specific gravity (relative density), Color, Appearance, pH value, Nitrite, Protein, Glucose, Ketones , Urobilinogen, Bilirubin, Erythrocytes, Leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes, Functional Observational Battery (Screen)
- Time schedule for examinations: last week of treatment
- Dose groups that were examined: all groups
- Battery of functions tested: Grip Strength, Locomotor Activity
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
n.a.
Postmortem examinations (parental animals):
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 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
n.a.
Statistics:
The following statistical methods were used to analyze body weight, grip strength, locomotor activity, clinical laboratory data, ophthalmoscopy, organ weights and ratios as well as macroscopic findings:
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher's exact-test
Reproductive indices:
n.a.
Offspring viability indices:
n.a.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
CLINICAL SIGNS AND MORTALITY
There were two premature decedents:
One male of the 12.5 mg/kg bw/d dose group was found dead on day 80 of treatment. In the 2 - 3 days prior to its death, clinical signs included weakened condition, ruffled fur and reddish nasal secretion.
One female of the 200 mg/kg bw/d dose group was sacrificed for ethical reasons on day 51 of treatment. This female could not move its hind limbs. Because it was unable to reach the food trough or water bottle, it was removed from the study.
All other animals survived until scheduled necropsy.

Test item-related clinical signs were noted in both genders at 200 mg/kg bw/d on the first day of treatment.
Tachypnea was noted in two males and one female, whereas convulsions and/or shivering were noted in three males and two females. Prostration, hunched posture, ruffled fur and/or stiff gait were noted in three females. Pica was also observed in four males and one female at the highest dose level. With the exception of a few transient incidents, these signs were no longer seen after day 1. Because the findings were of limited incidence and brief duration, they were considered to indicate acute toxicity from a metabolic overload which triggered a subsequent adaptive response.
Salivation, noted from the third treatment week until the end of treatment, is a typical finding of discomfort noted in rats treated with a test item formulation of unpleasant taste.
At 50 mg/kg bw/day, the acute toxic response was not evident. Salivation began during treatment week 3 and persisted until the end of treatment.
At 12.5 mg/kg bw/day, intermittent findings included pica, ruffled fur, salivation, and reddish nasal secretion (typically porphyrin).

Incidental findings included weakened condition, areas of hair loss, localized scabbing, kinked tail and/or chromodacryorrhea) were noted infrequently (including one control female) and were therefore considered to be of no relevance. One control female had a palpable mass on the left shoulder in week 13 of treatment.
No late effects of toxicological relevance were noted during the recovery period.

BODY WEIGHT AND FOOD CONSUMPTION
The mean absolute and relative body weights of the test item treated rats were similar to those of the controls. No late effects were noted during the recovery period.

ORGAN WEIGHTS
Test item-related differences in organ weights included significantly higher mean relative kidney weights in males at 50 mg/kg bw/day (p<0.05) and 200 mg/kg bw/day (p<0.01), and lower mean absolute thymus weight (p<0.01), mean thymus-to-body weight ratio (not significant) and mean thymus-to-brain weight ratio (p<0.05) in females at 200 mg/kg bw/day.
However, these differences did not correlate with microscopical changes of toxicological relevance.

GROSS PATHOLOGY
There were no test item-related adverse macroscopical findings at any dose level. A small number of typical background macroscopical findings were noted in males and females of the control and high-dose groups. The observed findings were either also (or only) seen in control animals, or only in individual animals of the high-dose group.
Male no. 22 (12.5 mg/kg bw/day), found dead on day 80 of treatment, was autolytic and no macroscopical findings could be recorded. Female no. 97 (200 mg/kg bw/d) was sacrificed for ethical reasons on day 51 of treatment. No macroscopical findings were evident.

HISTOPATHOLOGY
Treatment related microscopic findings were recorded in the stomach. At the end of the main study and in unscheduled deaths these findings consisted of:
At the end of the main study and in the two unscheduled deaths treatment related microscopic
findings consisted of:
Stomach
- forestomach erosion at slight degree was noted in one group 4 (200 mg/kg bw/day) male and at minimal degree in one group 4 (200 mg/kg bw/day) female.
- minimal forestomach ulceration was recorded in one group 4 (200 mg/kg bw/day) female
- forestomach squamous hyperplasia at minimal or moderate degree in two group 4 (200 mg/kg bw/day) males and in females at minimal degree in one group 2 (12.5 mg/kg bw/day), two group 3 (50 mg/kg bw/day) and seven group 4 (200 mg/kg bw/day) animals

Following the four week recovery period, minimal forestomach squamous hyperplasia remained in evidence in one female previously treated with 200 mg/kg bw/day. These findings are indicative of local irritation in the forestomach.
The remaining microscopic findings were within the range of background pathology encountered in Wistar rats at these ages in this type of study and occurred at similar incidences and severity in both control and treated rats.



Further details are given in the IUCLID section "Repeated dose toxicity".
Dose descriptor:
NOEL
Remarks:
fertility effects
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No adverse test substance-related effects to the reproductive organs were found for any parameter measured, especially regarding organ weights of ovary and testes and histopathology of gonads, uterus, mammary gland, prostate and seminal vesicle.
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: clinical signs
Remarks on result:
not measured/tested
Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
n.a.
Remarks on result:
not measured/tested
Remarks on result:
not measured/tested
Reproductive effects observed:
no

No adverse test substance-related effects to the reproductive organs were found for any parameter measured, especially regarding organ weights of ovary and testes and histopathology of gonads, uterus, mammary gland, prostate and seminal vesicle.

Conclusions:
The results from the evaluation of reproductive organs, especially organ weights of ovary and testis and histopathology of gonads from this 90 day rat gavage study revealed no indications of any substance-related effects up to and including the highest test dose of 200 mg a.i./kg bw/d.
Executive summary:

In a subchronic toxicity study according to OECD Guideline 408 (21 September 1998) and EU Method B.26 (30 September 1996 ), PETMP (97.4% a.i.) was administered to 10 RccHan: WIST(SPF) rats/sex/dose in corn oil by gavage at dose levels of 0, 12.5, 50 and 200 mg a.i./kg bw/day (corrections were made for purity) for 91 or 92 days.Additional animals in satellite groups (control and high dose, 5 males and 5 females, each) were kept for further 28 days without treatment to detect recovery from, or persistence of, toxic effects.

Test item-related findings were noted in some animals on the first day of treatment: tachypnea, convulsions and/or shivering, prostration, hunched posture, ruffled fur and/or stiff gait. Pica, atypical protective behavior for rats, was observed at this dose. These signs were generally seen only on day 1. The findings were of limited incidence and transient, and were considered to indicate acute toxicity from a metabolic overload which triggered a subsequent adaptive) if nonspecific) metabolic response. Findings of toxicological relevance were not seen at 50 mg/kg bw/day or 12.5 mg/kg bw/day.

There were no test item-related deaths, no differences in mean food consumption or body weights, weekly observations (weeks 1 - 13) or functional observational battery (week 13), no differences of toxicological relevance in the fore- and hind limb grip strength values, no test item-related differences in the ophthalmoscopy, no test item related effects on hematology or urine parameters. There were no test item-related macroscopic findings.

Test item-related differences in organ weights included higher mean relative kidney weights in males at 50 mg/kg bw/day and 200 mg/kg bw/day, and lower mean absolute thymus weight, mean thymus-to-body weight ratio and mean thymus-to-brain weight ratio in females at 200 mg/kg bw/day. However, these differences did not correlate with microscopical changes of toxicological relevance.

Test item-related microscopic findings were recorded in the stomach. At the end of the main study and in the two unscheduled deaths, these findings consisted of forestomach erosion (slight in degree) in one male at 200 mg/kg bw/day male and in one female at 200 mg/kg bw/day, forestomach ulceration (minimal in degree) in one female at 200 mg/kg bw/day, and forestomach squamous hyperplasia (minimal or moderate in degree) in two males at 200 mg/kg bw/day. This latter finding (minimal in degree) was also observed in one female at 12.5 mg/kg bw/day, two females at 50 mg/kg/day) and seven females at 200 mg/kg bw/day. Minimal forestomach squamous hyperplasia persisted after the recovery period in one female previously treated with 200 mg/kg bw/day. These findings are indicative of local irritation in the forestomach.

Based on these results, a no-observed-effect level (NOEL) for general toxicity could not be identified, and a specific target organ was not apparent. The LOEL for local effects (forestomach squamous hyperplasia) was 12.5 mg/kg bw/day. The LOEL for local effects, based on local irritation at the site of application (forestomach), is judged as not relevant to humans due to significant different anatomic situation and exposure probability in humans.

The no-observed adverse effect level (NOAEL) for general toxicity relevant to human DNEL calculation was considered to be 50 mg/kg bw/day.

No adverse test substance-related effects to the reproductive organs were found for any parameter measured, especially regarding organ weights of ovary and testes and histopathology of gonads, uterus, mammary gland, prostate and seminal vesicle. Thus, the NOEL related to fertility is considered to be 200 mg a.i./kg bw/d.

Reason / purpose:
read-across source
Reference
Endpoint:
fertility, other
Remarks:
assessment of reproductive organs in short term repeated dose toxicity study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5-7 weeks
- Weight at study initiation: Males - 134-179 g and females - 124-156 g
- Fasting period before study: No
- Housing: housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 December, 1999 To: 05 January, 2000
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The test substance was administered daily, for up to 28 d, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg bw/day of distilled water.
Details on mating procedure:
animals were not mated
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the test samples were done once per week for the entire duration of the study. The test substance formulations were diluted with methanol to give a final, theoretical test material concentration of approximately 0.1 mg/mL. Standard solutions of test substance were prepared in methanol at a nominal concentration of 0.1 mg/mL. For homogeneity determination, the test substance formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container. Sampling was performed in triplicate. For stability determination, the test substance formulations were sampled and analysed initially and then after storage at ambient temperature in the light for four hours. For analysis of concentrations, the test substance formulations were sampled and analysed immediately after preparation.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Details on study schedule:
animals were not mated
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Animals treated with 500 mg/kg bw/day showed a marked deterioration in
health and it was decided to stop dosing on Day 6. Dosing restarted on Day 7 at the revised dose level of 250 mg/kg bw/day and continued to the remainder of the study period.
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses for the main study were selected based on a 8-day dose range finding toxicity study in rats at 0, 150 and 500 mg/kg bw/day. No abnormalities were observed in this study and hence the doses for the main study were selected to be 0, 50, 150 and 500 mg/kg bw/day.
- Rationale for animal assignment (if not random): Randomised
- Rationale for selecting satellite groups: Based on standard practices, the recovery groups were selected to be vehicle control and high dose group.
- Post-exposure recovery period in satellite groups: 14 d
Parental animals: Observations and examinations:
Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all surviving non-recovery group animals at the end of the treatment period and for all surviving recovery group animals at the end of the treatment-free period.
Litter observations:
n.a.
Postmortem examinations (parental animals):
At the end of the dosing period, all animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.
Postmortem examinations (offspring):
n.a.
Reproductive indices:
n.a.
Offspring viability indices:
n.a.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs developed in the high dose animals on Day 2 of the study with a number of animals showing a severe deterioration in condition. Clinical signs among these animals included clonic convulsion, distended abdomen, dehydration, pallor of the extremities, hunched posture, lethargy, pilo-erection, decreased respiratory rate, gasping, laboured and noisy respiration and tiptoe gait. Following reduction of the dose level to 250 mg/kg bw/day on Day 7 clinical observations persisted throughout the treatment period and included distended abdomen, dehydration, hunched posture, pilo-erection, decreased, gasping,laboured and noisy respiration, increased salivation after dosing, red/brown staining of the mouth and snout, tiptoe gait. No clinical signs were observed in other dose levels.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were five treatment-related deaths during the study. One high dose male and two high dose female animals were killed in extremis on Day 5 of the study following a severe deterioration in condition. One high dose male was killed in extremis on Day 8 whilst a further male from this dose group died prior to treatment on Day 11. No mortality was observed from any other dose levels.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight gain was reduced at the high dose level during the first week of study and later recovered as the high dose was reduced to 250 mg/kg bw/day. No change in body weight was observed in other dose levels.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Similar to the effect on body weight gain, reduction in food consumption was observed at the high dose level which resolved from week 2.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Reductions in haemoglobin, haematocrit, erythrocyte count and mean corpuscular volume have been observed at the high dose and recovery groups. No effects were observed in other groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Reductions in total plasma protein, triglyceride, cholesterol and albumin were observed at the high dose. No changes in other groups.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Open-field assessments conducted during the study confirmed the clinical signs observed in high dose animals (500/250 mg/kg bw/day) throughout the study period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
Key result
Dose descriptor:
NOEL
Remarks:
fertility effects
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no effects to the reproductive organs
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Remarks on result:
not measured/tested
Remarks on result:
not measured/tested
Key result
Reproductive effects observed:
no
Conclusions:
Under the study conditions, the repeat dose toxicity NOAEL of the substance in rats was determined to be 150 mg/kg bw/day. The NOEL(fertility parameters) was 250 mg/kg bw/d.
Executive summary:

A study was conducted to determine the repeat dose toxicity of the test substance in rats according to OECD Guideline 407 and EU Method B.7, in compliance with GLP. The test substance was administered as an aqueous solution by oral gavage to groups of five male and five female Sprague-Dawley rats for 28 d, at dose levels of 0, 15, 150 and 500 mg/kg bw/day (incorporating a correction factor for 98.4% purity). Due to a deterioration in the health of the animals at 500 mg/kg bw/day, the highest dose level (including recovery group animals) was reduced to 250 mg/kg/day from Day 7. Treatment-related mortality, clinical signs, reduction in body weight gain and food consumption, reduction in red blood cell parameters and changes in clinical chemistry were observed at the high dose only. At gross pathology, treatment-related lesions in the stomach were observed in the high dose animals which died during the study. Under the study conditions, the repeat dose toxicity NOAEL of the substance in rats was determined to be 150 mg/kg bw/day. Based on no effects observed to the reproductive organs (organ weights (ovaries, epididymides and testes) and histopathology (ovaries, uterus,epididymides, prostate, seminal vesicles and testes)), the NOEL(fertility parameters) was 250 mg/kg bw/d.

Reason / purpose:
read-across source
Reference
Endpoint:
fertility, other
Remarks:
fertility parameters from 13 wk repeated dose toxicity study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study design states it complies with OECD guideline 408 (1981) and it follows latest OECD guideline 408 (1998) with deviations: no data on sensory reactivity to stimuli of different types, assessment of grip strength, and motor activity; blood clinical chemistry did not include urea; urinalysis did not include osmolality or specific gravity.
Qualifier:
according to
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1981
Deviations:
yes
Remarks:
No data on sensory reactivity to stimuli of different types, assessment of grip strength, and motor activity; blood clinical chemistry did not include urea; urinalysis did not include osmolality or specific gravity.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Sprague Dawley Ico:OFA-SD (lOPS Caw)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: IFFA CREDO, L'Arbresle, FRANCE.
- Age at study initiation: 6 weeks
- Weight at study initiation: 162 - 193 g (males) and 142 - 180 g (females)
- Housing: Groups of 5 of the same sex and dose group in stainless steel mesh cages (555 x 350 x 200 mm)
- Diet (e.g. ad libitum): Ad libitum sterile Rat and Mouse pelleted complete diet (Diet A04C-10, Usine d'Alimentation Rationnelle, Epinay S/Orge, France)
- Water (e.g. ad libitum): Ad libitum filtered (0.2 µm) mains water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 35 - 75
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
other: 1% Carboxymethylcellulose in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: the test article was prepared as a suspension in the vehicle

VEHICLE
- Concentration in vehicle: 12.5, 35, and 100 mg/mL
- Amount of vehicle (if gavage): 10 ml
Details on mating procedure:
animals were not mated
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For day 1, it was used a Varian 9010 high-performance liquid chromatograph equipped with a 9095 autosampler and a Kontron 432 UV detector.
For weeks 4, 8, and 13, it was used a Shimadzu LC 6A high-performance liquid chromatograph equipped with a SIL 6A autosampler and a UV detector SPD 6A.
Column Nucleosil C18, 120 x 4.6 mm I.D., particle size 3 μm; flow rate at 1 mL/min.
Detection at 209 nm.
Injection volume: 15 μL of each sample.
Mobile phase: solvent A: acetonitrile; solvent B: methanol isocratic mode : 60 % (A) and 40 % (B).
Samples (2 x 2 g) from each preparation (including the control group) were taken at weeks 1, 4, 8, and 13 and were stored at approximately -20 °C.
Duration of treatment / exposure:
Males: 92 days; females: 93 days; recovery animals: 91 days (plus treatment-free recovery period of 4 weeks)
Frequency of treatment:
Once daily
Details on study schedule:
animals were not mated
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
350 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 in each dose group of main study and 5 in recovery group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected based on the results of a dose range-finding study in the rat (study number 380/572). The high dose of 1000 mg/kg/day is the maximum required by the relevant Regulatory Guidelines, 125 mg/kg/day was the low dose level in the dose range-finding study and was expected to be a no-effect or no observable adverse effect level. The intermediate dose is an approximation of the geometric mean between the low. and high dose levels.
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, at the beginning and at the end of each working day
- Cage side observations: Morbidity/mortality, clinical signs, and reaction to the treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretest and week 13
- Dose groups that were examined: all animals pretest, oil animals during week 13 in control and high-dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: control and high-dose group after week 4, all groups after week 13, and recovery animals after week 17
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 10 animals/sex/group. All recovery animals after week 17
- Parameters checked: Haemoglobin; Mean corpuscular haemoglobin (MCH); Mean corpuscular haemoglobin concentration (MCHC); Packed cell volume; Red blood cell count; Mean corpuscular volume (MCV); Platelet count; Prothrombin time; Activated partial thromboplastin time (APTT); Total white blood cell count; Differential white blood cell count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: control and high-dose group after week 4, all groups after week 13, and recovery animals after week 17
- Animals fasted: Yes
- How many animals: 10 animals/sex/group. All recovery animals after week 17
- Parameters checked: Sodium; Potassium; Chloride; Calcium; Inorganic phosphorus; Glucose; Blood urea nitrogen; Total cholesterol*; Total bilirubin; Total protein; Albumin; Globulin (calculated); Albumin/Globulin ratio; Creatinine; Alkaline phosphatase (AP)*; Aspartate aminotransferase (ASAT)*; Alanine aminotransferase (ALAT)*.
*Only these parameters were examined after week 17 ; total cholesterol was evaluated in females only.

URINALYSIS: Yes
- Time schedule for collection of urine: control and high-dose group after week 4, all groups after week 13, and recovery animals after week 17
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes. Animals deprived of food and, water received 20 mL/kg of mains water by gavage before being placed in the metabolism cages
- Parameters checked: Volume; Specific gravity; Appearance. Semi-quantitative estimation: pH*; Protein; Glucose; Ketones; Urobilinogen; Bilirubin; Blood; Reducing substances; Microscopic examination of spun deposit
*Only this parameter was examined after week 17.
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
n.a.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes. All animals were submitted to full necropsy procedures including examination of: the external surface,all orifices, the cranial cavity, the carcass, the external surface of the brain and of samples of the spinal cord (spinal cord was examined at the time of tissue processing for animals examined histopothologically), the thoracic, abdominal and pelvic cavities and viscera, the cervical tissues and organs.
HISTOPATHOLOGY: Yes. The following organs/tissues were sampled for all animals: adrenals; aorta; bone (femur)* and articulation*; bone (sternum) with bone marrow; bone marrow smears; bronchi (mainstem); brain; cecum; colon; duodenum; epididymides; eyes; heart; ileum; jejunum; kidneys; lachrymal gland*; liver; lungs; lymph node (submaxillary); lymph node (mesenteric); mammary gland; esophagus; optic nerves; ovaries; pancreas; parathyroids; pituitary; prostate; rectum; salivary gland (submaxillary); sciatic nerve; seminal vesicles; skeletal muscle * (quadriceps femoris); skin; spinal cord (cervical, thoracic, lumbar); spleen; stomach (fundus, pylorus); testes; thymus (where identified); thyroids; trachea; urinary bladder; uterus (horn + cervix); all gross lesions;
* fixed but not processed initially
Postmortem examinations (offspring):
n.a.
Reproductive indices:
n.a.
Offspring viability indices:
n.a.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
HAEMATOLOGY. After 4 weeks of treatment, the mean platelet count of high dose males was statistically significantly higher than the control mean (p < 0.05). However, in the absence of any statistically significant difference in females at the some stage of the study, or in any other treatment group at the end of the treatment period, the toxicological significance of this small change was considered doubtful.
The mean activated partial thromboplastin time (APTT) of high dose males was also higher than the control mean at the week 4 sampling, but this was almost entirely due to animal n° 39. The reason for this high value is unclear, but because of the isolated nature of the change, it is considered to be unrelated to treatment.
At the end of the treatment period (week 13), high dose males had higher haemoglobin concentrations (Hb), packed cell volumes (PCV), and red blood cell counts (RBC) than respective controls. These differences were statistically significant (p < 0.05 and p < 0.001).
It should be borne in mind, however, that all the individual values in the high dose group for these parameters were within the extreme normal background range of the testing facility (with the exception of the PCV for animal n° 44) and that the control means for Hb and PCV were lower than the normal background mean.
In females at the end of the treatment period, all treated groups tended to have higher Hb concentrations than controls, although the difference was very small in intermediate dose females. In addition, high dose females had higher mean cell haemoglobin values than controls. These differences were statistically significant. In the absence of any dose-relationship and since the individual values fell consistently within the normal background range, the changes were considered unlikely to be a direct toxic effect of the test article.
A small number of high dose animals had slightly higher absolute or percentage neutrophil counts than controls at the end of treatment resulting in the difference between the means being statistically significant (p < 0.05). Since the majority of the individual values were within the normal background range, and based on the low incidence of this change, its toxicological relevance was considered to be doubtful.

CLINICAL CHEMISTRY. In general, high dose animals tended to have higher serum potassium, calcium, and phosphorus concentrations than controls at the end of the treatment period. In addition, the calcium concentrations of intermediate dose males were also slightly elevated compared with controls. However, the differences were small, often not dose-related and generally well within the normal background range. They were therefore considered to be of doubtful toxicological significance.
At the end of the treatment period, the mean cholesterol concentration of high dose females was higher than the control mean (p < 0.01). However, only three individual values fell at or outside two standard deviations of the background mean. When this parameter was measured at the end of 4 weeks without treatment there was no difference between the controls and those females previously treated at the high dose level, thus indicating that this small change was not a direct toxic effect of the test article.
High dose animals had higher serum alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activities than controls at the end of 13 weeks of treatment. There was also some evidence for a similar but much smaller effect on ALAT in intermediate dose females (p < 0.05). However, the magnitude of the difference was not large and the individual values were within the normal background range. In addition, a change in this parameter is not indicative of an effect on the heart. Consequently, the change was considered to be incidental to treatment. There were no differences between control animals and those previously treated at the high dose level when the parameters were measured in week 17.
None of the other small differences between treated and control groups, although occasionally reaching the level of statistical significance, was considered to represent a direct toxic effect of the test article.

URINALYSIS. Although the urine of high dose animals was slightly more acidic than that of the controls after 13 weeks of treatment when the pH of the urine was measured at the end of the treatment-free period there was no meaningful difference in the acidity of the urine collected from control animals and that collected from animals previously treated at the high dose level. There were no other differences in the urine parameters to indicate any effect of the test article.

ORGAN WEIGHTS. When compared with the control group, the mean liver weight was higher for high dose group males when expressed relative to body weight and for females when expressed in absolute terms and relative to body weight. In addition, when compared with controls, the mean testis weight was lower for high dose group (absolute and relative to brain weight).
High dose group females also had a lower mean ovarian weight when expressed relative to brain weight. When compared with the control group, the mean liver weight was higher for high dose group males when expressed relative to body weight and for females when expressed in absolute terms and relative to body weight. In addition, when compared with controls, the mean testis weight was lower for high dose group (absolute and relative to brain weight). High dose group females also had a lower mean ovarian weight when expressed relative to brain weight. In the absence of any microscopic lesions in these organs the weight changes were considered to be of doubtful toxicological significance.
At the end of the treatment-free period, the mean absolute brain weight for high dose group males was higher than the control mean and this group also had a statistically significantly higher mean (absolute and relative to brain weight) prostate and epididymidis weight than the control. In the absence of any microscopic lesions in these organs the weight changes were considered to be of doubtful toxicological significance. In the absence of any microscopic lesions in these organs, the weight changes were considered to be of doubtful toxicological significance.
Historical control data for organ weights were not given in the report.

GROSS PATHOLOGY. Most tissues were unremarkable at necropsy. The most common finding was pale area(s) on the stomach mucosa. The incidence of this finding was higher for high dose group males and females, but was also seen in the control group. Alopecia seen in high dose group males female was also seen in control females killed at the end of the treatment-free period and it was considered to be of no toxicological significance. Other macroscopic changes were considered to be either agonal or incidental in origin.

HISTOPATHOLOGY: NON-NEOPLASTIC. Treatment-related findings were seen in the heart. The lesion which was seen in intermediate dose group males and in high dose group males and females, was described as small foci of degenerated or necrotic fibers associated with minimal to moderate mononuclear cell infiltration. This association suggested early or on-going myocarditis. In high dose group males and females the lesions involved the right and/or the left ventricular wall and occasionally the interventricular septum. Such findings were multifocal and slightly more severe in males than in females. In intermediate dose group males, the lesions were minimal or slight focal and most often located in the subendocardial zones, in the interventricular septum, or in the papillary muscles.
In one female (N° 78), the mononuclear cell infiltration was in the right and left ventricular wall and multifocal, but it was not associated with small foci of degenerate or necrotic fibers. No ventricular infiltration was seen in the other females nor in the males.
Inflammatory changes in control and low dose group males and females were few and they were also in the subendocardial zones, in the septum, or in the papillary muscles. No change was seen in the ventricular walls.
Special staining by phosphotungstic acid haematoxylin (PTAH) did not indicate any modification of the striation of the myocardial fibers in the areas next to the small foci of necrosis.
The above findings were clearly related to the test article in high dose group males and females. In addition, this finding was correlated in males with an increase in serum aspartate aminotransferase (ASAT) activity. The activity of this enzyme is known to increase in cases of slight myocardial necrosis. However, in intermediate dose group the focal distribution of the histological changes (very focal and most often in the septum) and the lower grade of severity of the lesions (minimal), suggested that such findings had a doubtful toxicological significance, particularly in the absence of elevated ASAT levels, and were most probably unrelated to the administration of the test article. It is known that small foci of necrosis and focal inflammation and fibrosis occur spontaneously in the septum or in the papillary muscles in young rats, probably as a result of focal ischemia, and that this change becomes more common with increasing age. No abnormality was seen in the heart at the end of the recovery period.
In the stomach, mucous adherence on the glandular mucosa was correlated with the macroscopic observation of "pale areas". In addition, two superficial erosions of the mucosa were seen, one in male N° 31 from the intermediate dose group and one in female N° 95 from the high dose group. This finding was most probably related to the administration procedure (gavage).
Other findings were few or those normally observed in rats and they were considered to be agonal or incidental in origin and unrelated to the administration of the test article. No change was seen in the organs for which the weight was modified.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Dose descriptor:
NOAEL
Remarks:
fertility parameters
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: no effects to the reproductive organs observed
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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:
not examined
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Remarks on result:
not measured/tested
Remarks on result:
not measured/tested
Reproductive effects observed:
no
Conclusions:
NOAEL for fertility parameters: 1000 mg/kg bw/d (no effects to reproductive organs observed (epididymides, mammary gland, ovaries, prostate, seminal vesicles, testes, uterus))
Reason / purpose:
read-across source
Reference
Endpoint:
fertility, other
Remarks:
fertility parameters from 28 d repeated dose toxicity study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Tif:RAIf
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal Production, ClBA-GEIGY Limited, 4332 Stein / Switzerland
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 165.9-191.4 g (males), 159.6-183.9 g (females)
- Housing: in groups of 5 in macrolon IV cages
- Diet (e.g. ad libitum): Pelleted, certified standard diet Nafag No. 890 Tox, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±10
- Air changes (per hr): 16-20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: 0.5% CMC and 0.1% Tween 80 in distilled water
Details on exposure:
VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw
Details on mating procedure:
animals were not mated
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Control analyses of che test article concentration in the vehicle were carried out at all dose levels on samples collected once per experimental week. The samples were collected after administration, immediately deep frozen and sent to the analytical laboratories.
Duration of treatment / exposure:
28 d + 2 wk recovery
(800 mg/kg bw/d dose group was terminated on day 9)
Frequency of treatment:
daily
Details on study schedule:
animals were not mated
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 (5 for main group, 5 for recovery group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on acute oral toxicity study and further 28 d toxicty study (0, 2, 10, 50, 200 mg/kg bw/d)
Positive control:
no
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Time schedule: weekly, cagewise

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly, cagewise

OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment/recovery period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 10 (main group), 5 (recovery group)
- Parameters: erythrocyte count, hemoglobin, hematocrit, MCV, MCH, leukocyte count, differential leukocyte count, thrombocyte count, prothrombin time, methemoglobin

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood: end of treatment/recovery period
- Animals fasted: Yes
- How many animals: 10 (main group), 5 (recovery group)
- Parameters: glucose, urea, creatinin, total bilirubin, total protein, albumin, globulins, A/G ratio, cholesterol, triglycerides, sodium, potassium, calcium, chloride, anorganic phosphorus, ASAT, ALAT, ALP, GGT

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
no
Sperm parameters (parental animals):
not examined
Litter observations:
n.a.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
weight: brain, liver, kidneys, adrenals, ovaries/testes
preservation: skin, mammary area, spleen, mesenteric lymph node, axillary lymph node, popliteal lymph node, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submndibular salivary gland (both), liver, pancreas, esophagus, stomach, small intestine, large intestine, kidney (both), urinary bladder, prostate, seminal vesicle, testis (both), epididymis (both), uterus, vaglna, ovary (both), pituitary gland, adrenal gland (both), thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve (both), orbital gland (both), extraorbital lacrimal gland (both), Zymbal gland (both), muzzle, tongue, any tissue with gross lesions

HISTOPATHOLOGY: Yes
spleen, heart, liver, kidney, adrenal gland, any tissue with gross lesions
Postmortem examinations (offspring):
n.a.
Reproductive indices:
n.a.
Offspring viability indices:
n.a.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In 9/10 males and 8/10 females of group 5 (800 mg/kg) a variety of signs of toxicity were observed, including laboured breathing, bradypnoea, apathy, hump-backed posture, lateral and ventral position, lame hindquarters, piloerection, emaciation and priapism. From these animals, one male and 3 females were found dead before moribund sacrifice of the surviving animals from group 5 at experimental day 9.
No relevant symptoms or signs of toxicity were noted in the other treated groups.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
2 males and 5 females of the high dose group (800 mg/kg) were found dead between experimental days 2-6. Due to this mortality and marked signs of toxicity, the surviving animals treated at 800 mg/kg were sacrificed early at day 9.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Before early sacrifice of animals from group 5 (800 mg/kg) at week 2, decreased mean bodyweight of these animals was already recorded at week 1.
The bodyweight development of the other treated groups was undisturbed by the treatment with the test article.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Before sacrifice at week 2, the mean food consumption of animals treated at 800 mg/kg was drastically reduced to levels of 5 and 13% of the controls in males and females, respectively.
The mean food consumption of the other treated groups was comparable to the control groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At week 1, markedly reduced mean water consumption was recorded for animals of group 5 (800 mg/kg), which in moribund condition were sacrificed at week 2.
Compared to the controls, no relevant differences in water consumption were noted for the other treated groups.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A minimally higher number of white blood cells occurred in males and females of group 4 (200 mg/kg) at the end of the treatment period.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No effects on blood chemistry parameters attributable to the treatment were observed. All the minor differences between treated and control groups attaining a level of statistical significance represent the normal
physiological variation of the respective parameters, and are therefore not considered treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment related changes occurred only in group 5 (800 mg/kg):
6/10 males (no. 41, 43, 44, 45, 47 and 48) showed hyperplasia of the epithelium in the non-glandular stomach. accompanied in some cases by submucosal oedema. In 1/10 males (no. 46) the non-glandular stomach was ulcerated. 7/10 males (no. 41, 42, 43, 44, 46, 47 and 49) showed slight, moderate or marked fatty change of the perilobular region of the liver.
3/10 males (no. 43, 44 and 45) had slight or moderate tubular atrophy of the testis, accompanied in two cases by absence of spermatozoa in the epididymis.
4/10 males (no. 41, 42, 43 and 44) showed slight or moderate atrophy of the thymus.
4/10 females (no. 93, 94, 95 and 97) showed hyperplasia of the epithelium in the non-glandular stomach and in 3/10 females (no. 91, 92 and 93) from this group the non-glandular stomach was ulcerated.
8/10 females (no. 91, 92, 93, 95, 96, 97, 98 and 99) had slight, moderate or marked fatty change mainly of the perilobular region of the liver.
6/10 females (no. 91, 92, 93, 94, 95 and 97) showed slight, moderate or marked atrophy of the thymus.
In conclusion only the liver was regarded as the tarqet organ.
The other affected organs namely the stomach, testis/epididymis and thymus were thought to be damaged as the result of debilitating effect of systemic tOXicity and/or local irritation.
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Dose descriptor:
NOAEL
Remarks:
fertility parameters
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no effects to the reproductive organs observed
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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:
not examined
Histopathological findings:
not examined
Remarks on result:
not measured/tested
Remarks on result:
not measured/tested
Reproductive effects observed:
no
Conclusions:
Under the conditions of this test, no effects to the reproductive organs were noted in dose levels up to 200 mg/kg bw/d.
Executive summary:

In the present study 10 males 10 females per dose group were administered Di-2-EHDTDG by gavage for 4 weeks at doses of 0, 10, 50 and 200 mg/kg bodyweight. Due to early sacrifice of animals from group 5 (800 mg/kg), treatment was terminated at day 9 for these animals. In groups 1-4, 5 animals per sex and group were sacrificed at the end of the treatment period, 5 animals per sex and group were kept for a 2-week recovery period before sacrifice.

Administered quantities of the test article suspension were adjusted daily to individual bodyweight.

The results of this study are summarised as follows:

 

Antemortem findings:

In 9/10 males and 8/10 females of group 5 (800 mg/kg bw/d) a variety of signs of toxicity were observed, including laboured breathing, bradypnoea, apathy, bump-backed posture, lateral and ventral position, lame hindquarters, piloerection, emaciation and priapism.

 

Mortality:

2 males and 5 females of the high dose group (800 mg/kg bw/d) were found dead between experimental days 2-6. Due to mortality and marked signs of toxicity, the surviving animals treated at 800 mg/kg were sacrificed at day 9.

 

Bodyweight:

Before sacrifice of animals from group 5 (800 mg/kg bw/d) at week 2, decreased mean bodyweight of these animals was already recorded at week 1.

The bodyweight development of the other treated groups was undisturbed by the treatment with the test article.

 

Food consumption:

Before sacrifice at week 2, the mean food consumption of animals treated at 800 mg/kg bw/d was drastically reduced.

The mean food consumption of the other treated groups was comparable to the control groups

 

Water consumption:

At week 1, markedly reduced mean water consumption was recorded for animals of group 5 (800 mg/kg bw/d).

Compared to the controls, no relevant differences in water consumption were noted for the other treated groups.

 

Hematology:

A minimally higher number of white blood cells occurred in males and females of group 4 (200 mg/kg bw/d) at the end of the treatment period.

 

Blood chemistry:

No effects on blood chemistry parameters attributable to the treatment were observed.

 

Organ weights:

The analysis of organ weights revealed no remarkable effects to the treatment.

 

Pathology:

The following changes occurred in animals of group 5 (800 mg/kg bw/d), which were regarded as treatment related: fatty change of perilobular region of the liver, hyperplasia and/or ulceration of non-glandular stomach, tubular atrophy of the testis and atrophy of the thymus.

Only the liver was regarded as the target organ. The remaining changes were thought to be the result of debilitating effect of systemic toxicity and/or local irritation.

No lesions were observed in the animals of the other dose groups, which survived the treatment period.

Especially, no effects to the reproductive organs (prostate, seminal vesicle, testes, epididymides, uterus, vagina, ovaries) were noted.

 

Under the conditions of this test, treatment with Di-2-EHDTDG resulted in pronounced signs of toxicity and mortality in animals treated with 800 mg/kg bw/d, leading to an early sacrifice of surviving

animals at week 2. In these animals decreased mean bodyweight was noted, in correlation to reduced food and water consumption.

Histopathology revealed liver as a target organ, besides secondary changes due to debilitating effects of systemic toxicity and/or local irritation.

The minor hematological finding noted in animals dosed with 200 mg/kg bw/d was reversible after the recovery period.

It can be inferred from the observations during the study that a NOEL for Di-2-EHDTDG when administered to rats by gavage for a period of 4 weeks is 50 mg/kg bw/d.

 

Data source

Materials and methods

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:
not measured/tested

Results: F1 generation

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
20 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOEL
Generation:
F2
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
mortality

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Executive summary:

With the exception of the mercaptan family, data on toxicity to reproduction and/or development are available for all subgroups.

 

No effects on mating, fertility, estrous cycle and sperm parameters were observed in any of the studies. There was no evidence of damage to the reproductive organs. Nor did any of the substances show developmental toxicity or teratogenicity.

 

The effects observed in the reproduction/developmental toxicity screening test and the 2-generation reproduction toxicity test with NaTG consisting of a significantly longer gestation period, a non-significantly lower number of corpora lutea, a significantly lower number of implantations and reduced pup survival were only seen in the presence on severe maternal toxicity including mortality and thus, secondary effects.

In a comparable manner, mean live litter size, postnatal survival and pup body weights and body weight gains were reduced in the high dose groups of the reproduction / developmental toxicity screening test with 2-EHTG in the presence of marked general toxicity (mortality).

 

Also, the increase in fetal variations observed in the dermal prenatal developmental toxicity test with NaTG (slightly increased incidences of extra or rudimentary ribs on Lumbar I and of dumbbell cartilage and bipartite ossification in thoracic centra - common skeletal variations in term rat fetuses), is interpreted as secondary to intrauterine growth retardation resulting from maternal toxicity, especially in combination with the absence of teratogenic alterations and with reductions in maternal and fetal weight.

 

Similar effects were seen for the members of the 3-MPA family. Lower mean male fetal weight, increased incidence of fetuses showing incomplete ossification of the thoracic centrum or less than four ossified caudal vertebrae, increased incidence of absent renal papilla were observed in the prenatal developmental toxicity study with PETMP in the high dose in the presence of marked general toxicity in dams.

 

TLA did not cause effects to the reproductive organs in a 28 d repeated dose toxicity study.

 

E12 caused no developmental toxicity in rat, mouse, hamster, and rabbit. Noeffects tothereproductive organswere noted in a 13 wk repeated dose toxicity study.Di-2-EHDTDG did not cause effects tothereproductive organsin a 28 d repeated dose toxicity study.

 

Overall, it can be stated, that this whole group of substances has a very low potential to cause reproductive or developmental toxicity. Effects (e.g., lower fetal weight, intrauterine growth retardation, reduced postnatal survival) were observed only in the presence of severe general maternal toxicity.

 

Based on the available data, this group of substances does not need to be classified for toxicity to reproduction or developmental toxicity.

 

For better comparison, the NO(A)ELs have been recalculated on the basis of S-content, which is assumed to be the main driver for toxicity.

 

Overall comparison of NO(A)ELs (toxicity to reproduction)

Family

Substance

(study)

NO(A)EL [mg/kg bw/d]

S in molecule [%]

Related to S-content [mg/kg bw/d]

TGA

ATG

(NOEL, OECD TG 414, oral: gavage, rat)

75

 

32.1

24.1

NaTG

(NOAEL, OECD TG 416, oral: gavage, rat)

20

30.7

6.1

GMT

(reproduction NOEL, OECD TG 422, oral, rat)

50

21.1

10.5

2-EHTG

(reproduction NOAEL, OECD TG 421, oral: gavage, general toxicity, rat)

50

17.1

8.6

3-MPA

MMP

(reproduction NOAEL, OECD TG 422, oral: gavage, rat)

100

29.1

29.2

PETMP

(fertility NOAEL, OECD TG 408, oral: gavage, rat)

200

7.2

14.3

PETMP

(developmental NOAEL, OECD TG 414, oral: gavage, rat)

120

 

7.2

8.6

TLA

TLA

(fertility parameters NOAEL, OECD TG 407, oral:gavage, rat)

250

33.0

82.5

Intra-molecular-S

E12

(developmental NOAEL, OECD TG 414, oral: gavage, rat, mouse, hamster, rabbit)

>/=1600

6.8

108.8

E12

(fertility parameters NOEL, OECD TG 408, oral:gavage, rat)

1000

6.8

68

Di-2-EHDTDG

(fertility parameters NOAEL, OECD TG 407, oral:gavage, rat)

200

9.3

18.7

 

After adjustment to S-content, the NOAELs of most of the substances were within the same order of magnitude. Using the lowest NOAEL obtained in the 2-generation reproduction toxicity study conducted with NaTG is considered to be an appropriate starting point for DNEL derivation. Remaining DNEL uncertainties due to read-across can be adequately considered by applying an additional assessment factor.