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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

The potential toxic effects of 2,2-Bis(methylthio)propane was evaluated following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until day 5 post-partum (p. p.) (Bentz, 2012). The study, performed following the OECD guideline No. 421 and the principles of Good Laboratory Practices, provides initial information on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition. Three groups of 10 male and 10 female Sprague-Dawley rats received 2,2-Bis(methylthio)propane, daily, by oral (gavage) administration, before pairing and through pairing and, for the females, through gestation until day 5p. p.2,2-Bis(methylthio)propane was administered as a solution in the vehicle, corn oil, at dose-levels of 40, 160 and 640 mg/kg/day. Another group of 10 males and 10 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 concentration of the dose formulations was checked at each preparation (thus three times during the study) before the administration to the animals. The animals were checked at least twice daily during the dosing period for mortality and morbidity and at least once daily for clinical signs. Body weight and food consumption were recorded once a week. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5p. p. The total litter sizes and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs, abnormal behavior and external abnormalities and weighed on days 1 and 5 p. p. The males were sacrificed after completion of the pairing period and the dams on day 6p. p. Final body weights and selected organs weights (epididymides, prostate, testes, kidneys, liver, spleen) were recorded and a macroscopicpost-mortemexamination of the principal thoracic and abdominal organs was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs (epididymides, ovaries, testes, kidneys, liver, spleen) from the control- and high-dose groups, on kidneys (males), spleen (females) and liver (both sexes) from the low- and intermediate-dose groups and on all macroscopic lesions. The pups were sacrificed on day 5 p. p.and submitted for a macroscopicpost-mortemexamination of the principal thoracic and abdominal organs.

2,2-Bis(methylthio)propane concentrations in the administered dose formulations analyzed at each preparation were within the acceptance criteria (± 10%). There were no 2,2-Bis(methylthio)propane -related deaths in parents. At 640 mg/kg/day, hypoactivity, loss of balance, staggering gait, hypotonia, locomotory difficulties and abdominal breathing were observed at the beginning of the treatment period. They reappeared towards the end of gestation and disappeared in the lactation period. All animals of that group were affected by at least one of these clinical signs, females more than males. Although these clinical signs lasted the first 4 days and reappeared at the end of gestation only, they were particularly strong and likely induced in one female an abnormal maternal behaviour leading to cannibalism of its entire litter. They were therefore considered as adverse at 640 mg/kg/day. At 160 mg/kg/day, there were also hypoactivity and mainly loss of balance in males and especially in females at the beginning of the treatment period only. These clinical signs were considered as non adverse at this dose-level as less severe. Ptyalism was recorded in a dose-related manner from 40 mg/kg/day in males or at 640 mg/kg/day in females and was considered not to be adverse. There were no 2,2-Bis(methylthio)propane-related effects on mean body weight and on mean food consumption at any dose-level. There were no test item-related effects on reproductive parameters (mating, fertility and delivery data). At necropsy, there were higher mean kidney and liver weights in both sexes at 640 mg/kg/day and higher mean kidney weights in males at 160 mg/kg/day. There were also higher mean spleen weights in females at 160 and 640 mg/kg/day. Liver and kidneys were enlarged in three and two animals respectively at 640 mg/kg/day and tan discoloration of the kidney was also noted in three males. At microscopic examination at 640 mg/kg/day, there were centrilobular hepatocellular hypertrophy in all males and females, along with minimal vacuolation in two males and pigment-laden Küpffer cells in seven females. Moderate to marked tubular hyaline droplets were noted in the kidneys of all males, with foci of tubular basophilia (minimal to slight; occasionally with degenerated cells). At this dose-level, there was also minimally increased extramedullary hematopoiesis in the spleen of females. At 160 mg/kg/day, higher incidence and severity of tubular hyaline droplets were noted in the kidney of males, along with foci of tubular basophilia. Minimal centrilobular hepatocellular hypertrophy was also noted in 4/10 males. In the absence of associated microscopic degenerative changes, the liver changes are usually seen as an adaptative response and considered not to be adverse. In addition, the adverse kidney effects in the male rats were considered to be rat-specific and with no relevance for human risk assessment.

In pups, there were no 2,2-Bis(methylthio)propane-related deaths but cannibalism of one litter at 640 mg/kg/day may have been the consequence of the dam clinical condition at the beginning of the lactation period due to the test item treatment. At 640 mg/kg/day, three pups from three different litters were born with no or shortened tail. When compared with controls, there was a lower mean pup body weight on day 1p. p.in both sexes (6.4 and 6.2 g in males and females, respectively,vs.7.2 and 6.9 g in controls), reaching statistical significance for male pups (p < 0.05). Thereafter, the pups had a statistically significant retardation in mean body weight gain (+2.8 g in both sexes,vs.+4.1 and + 4.0 g in control males and females, respectively, p < 0.05). These effects on pup were considered to be test item-related and adverse. There were no test item-related effects on the pup sex ratio at any dose-level or on mean pup body weights and mean pup body weight gains at 40 and 160 mg/kg/day. There were no test item-related macroscopicpost-mortemfindings in pups at 40 and 160 mg/kg/day.

In conclusion, 2,2-Bis(methylthio)propane was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before pairing, during pairing, gestation and until day 5p. p.,at dose-levels of 40, 160 and 640 mg/kg/day.

Based on the experimental conditions of this study.

 the dose-level of 160 mg/kg/day was considered to be an Adverse Effect Level in rats as renal hyaline droplets were associated with tubular basophilia, suggesting previous chronic cell damage and increased cell turnover. However, as renal hyaline droplets are considered to be specific to male rats and therefore not relevant for human, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 160 mg/kg/day (because of the strong clinical signs and the effects on the female spleen seen at 640 mg/kg/day).

 the NOAEL for reproductive performance (mating and fertility) was considered to be 640 mg/kg/day.

 the NOEL for toxic effects on progeny was considered to be 160 mg/kg/day (based on the observation of pups with no or shortened tail and the effects seen on mean pup body weights and mean pup body weight gain at 640 mg/kg/day).

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 March 2012 - 09 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: the males were approximately 10 weeks old and the females were approximately 9 weeks old
- Mean body weight at study initiation: the males had a mean body weight of 347 g (range: 329 g to 367 g) and the females had a mean body weight of 221 g (range: 195 g to 253 g)
- Fasting period before study: no
- Housing: at receipt, the animals were housed by three or five from the same sex in suspended wire-mesh cages. A metal tray containing autoclaved sawdust (SICSA, Alfortville, France) was placed under each cage.
From randomization, the animals were individually housed, except during pairing and lactation, in Individually Ventilated Cages (IVC) (polysulfone 900 cm2, Tecniplast). Individual housing was chosen because of software limitations and since it is preferable for pregnant animals.
Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) was provided to females and their litters as nesting material.
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: : the animals were acclimated to the study conditions for a period of 7 days before the beginning of the treatment period.

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

IN-LIFE DATES: 22 March 2012 to 17 May 2012.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The Sponsor supplied the solutions prepared at the concentrations of 0 (corn oil), 8, 32 and 128 mg/mL intended for use in study groups 1, 2, 3 and 4, respectively. This preparation was carried out as follows: a total of 25 brown vials of at least 50 mL each, plus one vial of 200-250 mL, were prepared three times during the study (at a 3 week-interval approximately). The 25 vials were received by the labs on 20 March 2012, 11 April 2012 and 02 May 2012 for dosing of animals purposes. The remaining vials of 200-250 mL were kept by the sponsor to determine the concentration.
The density of formulation was not measured for safety precautions.
The dose formulations were received by the labs at room temperature and protected from light. From receipt, the formulation was stored at room temperature and protected from light for up to 3 weeks of use. Each eve of dosing, the volume required for the forthcoming day was stored in a safety cabinet in the animal unit, at room temperature and protected from light.
High precautions were taken to avoid the heating of the solutions.

VEHICLE
- Justification for use and choice of vehicle: test item soluble in corn oil
- Concentration in vehicle: 8, 32 and 128 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurs
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred as day 0 post-coitum
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: GC-FID
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: not assessed, dose formulation is a solution
Stability: stable for 4 weeks
Duration of treatment / exposure:
In the males:
- 2 weeks before mating,
- during the mating period,
- until sacrifice (i.e. at least 5 weeks in total),
In the females:
- 2 weeks before mating,
- during the mating period,
- during pregnancy,
- during lactation until day 5 post-partum inclusive,
- until sacrifice for females which had not delivered.
Frequency of treatment:
Daily
Details on study schedule:
- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: 11-12 weeks
Remarks:
Doses / Concentrations:
40, 160 and 640 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected by the Sponsor, following the results of a previous 4-week toxicity study.
In the study, the test item was given by gavage to Sprague-Dawley rats in corn oil at 80, 240 or 720 mg/kg/day for 4 weeks.
Ptyalism was observed at 720 mg/kg/day and minimal disturbances of red blood cell parameters suggesting increased red blood cell turn over were recorded in females given 720 or 240 mg/kg/day.
Treatment-related organ weight changes were recorded in the kidney of males treated from 240 mg/kg/day, the liver of males and females treated from 240 mg/kg/day and the spleen of females treated from 240 mg/kg/day.
Treatment-related lesions were observed in the kidneys of male rats from 80 mg/kg (eosinophilic hyaline droplets with tubular degeneration/necrosis), the liver (hepatocellular hypertrophy) and the spleen (extramedullar hematopoiesis or EMH) at 720 and/or 240 mg/kg/day. None of these morphological changes were considered as adverse. EMH and hemosiderosis was considered to be a compensatory effect, secondary to the test item-related effect on red blood cells.
Based on the aforementioned results, the high dose-level of 640 mg/kg/day was selected in order to produce signs of toxicity. The two others were selected according to a 4-fold interval.

- Rationale for animal assignment: computerized stratification procedure.
Positive control:
no (not required)
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: at least twice a day (mortality) or once a day (clinicals signs) during the treatment period.

BODY WEIGHT (GAIN):
- Time schedule: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mating (or until sacrifice), on Days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION:
- Time schedule:
The quantity of food consumed by each male was measured once a week, over a 7-day period, from the first day of treatment until the start of the pairing period.
The quantity of food consumed by each female was measured once a week, over a 7-day period, from the first day of treatment until the start of pairing period, during pregnancy at the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for interval days 1-5 p.p.
During the pairing period, the food consumption was not measured for males or females.
Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded.
Oestrous cyclicity (parental animals):
fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until females are mated.
Sperm parameters (parental animals):
Parameters examined in males of parental generation:
- testis weight (all groups) + microscopic evaluation (control and high-dose groups)
- epididymis weight (all groups) + microscopic evaluation (control and high-dose groups)
- microscopic evaluation of stages of the spermatogenic cycle and testicular interstitial cells (control and high-dose groups).
Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all animals after the end of the mating period
- Female animals: all surviving animals = day 6 post-partum or, for females which had not delivered yet, day 25 or 26 post-coitum (mothers with litter dying entirely were sacrificed as appropriate)

GROSS NECROPSY
Macroscopic post-mortem examination of principal thoracic and abdominal organs.

HISTOPATHOLOGY
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table from all animals of the control and high-dose groups (groups 1 and 4) sacrificed at the end of the treatment period , and for the prematurely female (Group 4) with total litter loss,
- all macroscopic lesions of all groups,
- on kidneys (males), spleen (females) and liver (both sexes) from animals of the low- and intermediate-dose groups (groups 2 and 3) sacrificed at the end of the treatment period.

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

ORGAN WEIGHTS: epididymes, prostate, testes, kidneys, liver and spleen.
Postmortem examinations (offspring):
SACRIFICE: on day 5 post-partum

GROSS NECROPSY: on all pups (surviving and found dead)

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No
Statistics:
Body weight, food consumption and reproductive data
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).
Organ weight
PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01).
Reproductive indices:
Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantations
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)
Offspring viability indices:
Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on day 4 p.p. = 100 * (Number of surviving pups on day 4 p.p. / Number of live born pups)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 640 mg/kg/day, the clinical signs (Tables 1 to 4), except ptyalism, were observed during the first 4 days of treatment. They reappeared in females towards the end of the gestation period and generally disappeared in the lactation period. Two females experienced again staggering gait and/or hypoactivity on day 5 p.p.
Although these clinical signs lasted 4 days only at the beginning of the treatment period and reappeared in pregnant females only at the end of gestation, they were particularly strong and likely induced in one female an abnormal maternal behaviour leading to cannibalism of its entire litter. They were therefore considered as adverse at 640 mg/kg/day.
Hypoactivity and loss of balance were also noted at 160 mg/kg/day for up to 4 or 5 days at the beginning of the treatment period.
As these clinical signs were less severe, they were considered as non adverse at this dose-level.
Ptyalism was recorded in a dose-related manner from 40 mg/kg/day in males or at 640 mg/kg/day in females and was considered to be non adverse.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
At 640 mg/kg/day, one female was sacrificed prematurely on day 3 p.p. because of the death of its litter.
There were no other unscheduled deaths during the study.
A total of five females (1, 2 and 2 given 40, 160 or 640 mg/kg/day, respectively) were sacrificed on day 25 or 26 p.c. for absence of delivery. All these females were non pregnant.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related effects on mean body weight and mean body weight gain.
In absence of any dose relationship, the statistically significant low mean body weight change in week 3 noted at 40 mg/kg/day in males was considered to be fortuitous.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item-related effects on mean food consumption.
There was a slightly lower mean food consumption noted at the beginning of the lactation period at 640 mg/kg/day (–11% vs. controls). This finding was considered to be of non toxicological significance (no statistical significance and low amplitude).
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Premature decent
Minimal centrilobular hepatocellular hypertrophy and moderate extramedullary hematopoiesis in the spleen were noted in the female treated at 640 mg/kg/day.
These microscopic findings were attributed to treatment with the test item.

Terminal sacrifice
Liver
Centrilobular hepatocellular hypertrophy was noted in all males and surviving females treated at 640 mg/kg/day, along with minimal vacuolation in 2/10 males and pigment-laden Kupffer cells in 7/9 females.
Minimal centrilobular hepatocellular hypertrophy was also noted in 4/10 males treated at 160 mg/kg/day.
In the absence of associated microscopic degenerative changes, this finding is usually seen as an adaptive response and considered not to be adverse.

Kidney
Increased incidence and severity of tubular hyaline droplets were noted in the kidneys of males treated at 160 or 640 mg/kg/day, along with foci of tubular basophilia (minimal to slight; occasionally with degenerated cells).

This correlated with increases in kidney weights and was characterized by the presence of dense eosinophilic droplets in proximal tubular epithelium. These hyaline droplets are consistent with a2µ globulin and are known to increase after treatment with a wide range of drugs or chemicals (Greaves, 2007). The prolonged accumulation of hyaline droplets is associated with chronic cell damage and increased cell turnover, which explains tubular basophilia seen in groups 3 and 4. These findings were therefore considered to be adverse. Although human excretes proteins of a similar nature, they are found in only trace amounts and therefore this finding is considered to be non-relevant for human.

Spleen
Minimally increased extramedullary hematopoiesis was observed in the spleen of females treated at 640 mg/kg/day.

Other microscopic findings were of those commonly reported in the rat and therefore a relationship to treatment was excluded.
More specifically, there were no evidences of effects in the testis or in the ovaries.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating and fertility data
There were no test item-related effects on mating or fertility data.
In the control group, there was one pair that mated after 13 days of pairing.
At 160 mg/kg/day, there was one pair that did not mate within 2 weeks. The female was blocked in diestrous for several days. Then, the female was paired with a second male and they mated the first night. Another pair in that group mated after 14 days of pairing (the female was blocked in diestrous and was non-pregnant).
These findings were recorded sporadically both in control- and test item-treated groups and therefore were considered not to be test item-related.
All the other pairs mated in similar pre-coital time.
There were 1, 2 and 2 non-pregnant females at 40, 160 and 640 mg/kg/day, respectively, which were sacrificed on day 25 or 26 p.c. due to no delivery. They had from 3 to 6 corpora lutea only at necropsy, vs. a range of 11 to 22 corpora lutea in pregnant females among all groups. A relationship with the treatment with the test item was considered to be doubtful in view of the similar pre-implantation loss among groups.

Delivery data
There were no test item-related effects on mean delivery data.
The slightly higher mean post-implantation loss noted at 40 mg/kg/day was due to one female which had 17 implants on the uterus but 6 live pups delivered only.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
reproductive performance (mating and fertility)
Effect level:
>= 640 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
yes
Lowest effective dose / conc.:
640 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no test item-related deaths in pups.
The lower viability index noted at 640 mg/kg/day was due to one found dead litter from one female which was cannibalized mainly on day 1 p.p. This dam had notably hypotonia and/or staggering gait when in presence of its litter, while the other females from this group usually did no longer have these clinical signs on days 1 and 2 p.p. The health condition was considered to be the cause of this abnormal maternal behavior and cannibalization.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no effects on mean pup body weight and mean pup body weight gain at the beginning of the lactation period at 40 and 160 mg/kg/day.
At 640 mg/kg/day, there was a lower mean pup body weight on day 1 p.p. in both sexes when compared with controls, reaching statistical significance in male pups. Thereafter, pups had a statistically significant lower mean body weight gain when compared with controls.
These findings were considered to be test item-related and adverse.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 640 mg/kg/day, three pups from three different litters had no tail (or shortened tail as described at necropsy). One of them, which also had a misshapen hindlimb, was cannibalized at the beginning of the lactation period.
This finding was not found in the other groups, is not commonly observed (for information: CIT Historical Control Data in embryo-fetal toxicity study: 1 acaudate fetus out of 120 litters) and was thus considered to be test item-related and adverse.
Histopathological findings:
not examined
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
gross pathology
Critical effects observed:
not specified
Reproductive effects observed:
yes
Lowest effective dose / conc.:
640 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before pairing, during pairing, gestation and until day 5 p.p., at dose-levels of 40, 160 and 640 mg/kg/day.
Based on the experimental conditions of this study:
- the dose-level of 160 mg/kg/day was considered to be an Adverse Effect Level in rats as renal hyaline droplets were associated with tubular basophilia, suggesting previous chronic cell damage and increased cell turnover. However, as renal hyaline droplets are considered to be specific to male rats and therefore not relevant for human, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 160 mg/kg/day (because of the strong clinical signs and the effects on the female spleen seen at 640 mg/kg/day),
- the NOAEL for reproductive performance (mating and fertility) was considered to be 640 mg/kg/day,
- the NOEL for toxic effects on progeny was considered to be 160 mg/kg/day (based on the observation of pups with no or shortened tail and the effects seen on mean pup body weights and mean pup body weight gain at 640 mg/kg/day).
Executive summary:

The potential toxic effects of Bismethylthiopropane was evaluated following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until day 5 post-partum (p.p.). The study, performed following the OECD guideline No. 421 and the principles of Good Laboratory Practices, provides initial information on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition. Three groups of 10 male and 10 female Sprague-Dawley rats received the test item, daily, by oral (gavage) administration, before pairing and through pairing and, for the females, through gestation until day 5 p.p. The test item was administered as a solution in the vehicle, corn oil, at dose-levels of 40, 160 and 640 mg/kg/day. Another group of 10 males and 10 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 concentration of the dose formulations was checked at each preparation (thus three times during the study) before the administration to the animals. The animals were checked at least twice daily during the dosing period for mortality and morbidity and at least once daily for clinical signs. Body weight and food consumption were recorded once a week. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs, abnormal behavior and external abnormalities and weighed on days 1 and 5 p.p. The males were sacrificed after completion of the pairing period and the dams on day 6 p.p. Final body weights and selected organs weights (epididymides, prostate, testes, kidneys, liver, spleen) were recorded and a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs (epididymides, ovaries, testes, kidneys, liver, spleen) from the control- and high-dose groups, on kidneys (males), spleen (females) and liver (both sexes) from the low- and intermediate-dose groups and on all macroscopic lesions. The pups were sacrificed on day 5 p.p. and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.

The test item concentrations in the administered dose formulations analyzed at each preparation were within the acceptance criteria (± 10%).

There were no test item-related deaths in parents. At 640 mg/kg/day, hypoactivity, loss of balance, staggering gait, hypotonia, locomotory difficulties and abdominal breathing were observed at the beginning of the treatment period. They reappeared towards the end of gestation and disappeared in the lactation period. All animals of that group were affected by at least one of these clinical signs, females more than males. Although these clinical signs lasted the first 4 days and reappeared at the end of gestation only, they were particularly strong and likely induced in one female an abnormal maternal behaviour leading to cannibalism of its entire litter. They were therefore considered as adverse at 640 mg/kg/day. At 160 mg/kg/day, there were also hypoactivity and mainly loss of balance in males and especially in females at the beginning of the treatment period only. These clinical signs were considered as non adverse at this dose-level as less severe. Ptyalism was recorded in a dose-related manner from 40 mg/kg/day in males or at 640 mg/kg/day in females and was considered not to be adverse. There were no test item-related effects on mean body weight and on mean food consumption at any dose-level. There were no test item-related effects on reproductive parameters (mating, fertility and delivery data). At necropsy, there were higher mean kidney and liver weights in both sexes at 640 mg/kg/day and higher mean kidney weights in males at 160 mg/kg/day. There were also higher mean spleen weights in females at 160 and 640 mg/kg/day. Liver and kidneys were enlarged in three and two animals respectively at 640 mg/kg/day and tan discoloration of the kidney was also noted in three males. At microscopic examination at 640 mg/kg/day, there were centrilobular hepatocellular hypertrophy in all males and females, along with minimal vacuolation in two males and pigment-laden Küpffer cells in seven females. Moderate to marked tubular hyaline droplets were noted in the kidneys of all males, with foci of tubular basophilia (minimal to slight; occasionally with degenerated cells). At this dose-level, there was also minimally increased extramedullary hematopoiesis in the spleen of females. At 160 mg/kg/day, higher incidence and severity of tubular hyaline droplets were noted in the kidney of males, along with foci of tubular basophilia. Minimal centrilobular hepatocellular hypertrophy was also noted in 4/10 males.  In the absence of associated microscopic degenerative changes, the liver changes are usually seen as an adaptative response and considered not to be adverse. In addition, the adverse kidney effects in the male rats were considered to be rat-specific and with no relevance for human risk assessment.

In pups, there were no test item-related deaths but cannibalism of one litter at 640 mg/kg/day may have been the consequence of the dam clinical condition at the beginning of the lactation period due to the test item treatment. At 640 mg/kg/day, three pups from three different litters were born with no or shortened tail. When compared with controls, there was a lower mean pup body weight on day 1 p.p. in both sexes (6.4 and 6.2 g in males and females, respectively, vs. 7.2 and 6.9 g in controls), reaching statistical significance for male pups (p < 0.05). Thereafter, the pups had a statistically significant retardation in mean body weight gain (+2.8 g in both sexes, vs. +4.1 and + 4.0 g in control males and females, respectively, p < 0.05). These effects on pup were considered to be test item-related and adverse. There were no test item-related effects on the pup sex ratio at any dose-level or on mean pup body weights and mean pup body weight gains at 40 and 160 mg/kg/day. There were no test item-related macroscopic post-mortem findings in pups at 40 and 160 mg/kg/day.

In conclusion, bis methylthiopropane was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before pairing, during pairing, gestation and until day 5 p.p., at dose-levels of 40, 160 and 640 mg/kg/day.

Based on the experimental conditions of this study:

. the dose-level of 160 mg/kg/day was considered to be an Adverse Effect Level in rats as renal hyaline droplets were associated with tubular basophilia, suggesting previous chronic cell damage and increased cell turnover. However, as renal hyaline droplets are considered to be specific to male rats and therefore not relevant for human, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 160 mg/kg/day (because of the strong clinical signs and the effects on the female spleen seen at 640 mg/kg/day),

. the NOAEL for reproductive performance (mating and fertility) was considered to be 640 mg/kg/day,

. the NOEL for toxic effects on progeny was considered to be 160 mg/kg/day (based on the observation of pups with no or shortened tail and the effects seen on mean pup body weights and mean pup body weight gain at 640 mg/kg/day).

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

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the available data, no classication for reproductive toxicity is warranted for 2,2-bis(methylthio)propane following Regulation (EC) No 1272/2008.

Additional information