Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 February 2009 - 13 October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guideline (deviations were not considered to have compromised the validity of the study and results); adequate coherence between data, comments and conclusions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
the temperature and the relative humidity were sometimes outside the target range + deviations to study plan
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
yes
Remarks:
idem above
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 440/2008 B.35
Deviations:
yes
Remarks:
idem above
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Sodium thioglycolate
- Physical state: violet liquid (aqueous solution of sodium thioglycolate)
- Analytical purity: 46% (for batch No. 26461) and 46.1% (for batch No. 25462) w/w (idometric)
- Purity test date: 05 May 2009 (for batch No. 26461) and 12 January 2009 (for batch No. 25462)
- Batch Numbers: 26461 and 25462
- Expiration dates of the batches: 05 July 2009 (for batch No. 25462) and 04 May 2010 (for batch No. 26461)
- Storage conditions of test material: in the original bottles, at ambient temperature and under nitrogen atmosphere.

Test animals

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.

Administration / exposure

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
Doses / concentrations
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.

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

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.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Remarks:
parental toxicity
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
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:
other: see 'Remark'
Dose descriptor:
NOEL
Remarks:
fertility and gestation
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Remarks on result:
other: Generation: P and F1 (migrated information)
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:
other: death of susceptible pregnant females around the time of delivery.
Remarks on result:
other: Generation: P and F1 (migrated information)
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
Remarks:
developmental toxicity
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
other: Generation: F1 and F2 (migrated information)
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: effects on the dam include lack of nesting/nursing behavior and this causes death of the pups which have been delivered
Remarks on result:
other: Generation: F1 and F2 (migrated information)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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.