2,2'-[1,2-ethanediylbis(oxy)]bis(ethanethiol)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The effects of the test item, DIMERCAPTO-1,8-DIOXA-3,6-OCTANE (DMDO), on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of conceptus, parturition and early lactation of the offspring were investigated, when administered orally to male and female Sprague Dawley SD rats. Groups of 10 males and 10 females received the test item by gavage at dosages of 50, 100 and 150mg/kg/day.The following investigations were performed on parental animals of all groups: mortality check, clinical signs, body weight, body weight gain, food consumption and mating per- formance, oestrous cycle evaluation for parental females (2 weeks before dosing, during pre-mating and mating phases, prior to necropsy), thyroid hormone determination only for parental males, litter data, macroscopic observations, organ weights and histopathological examination. Clinical signs, anogenital distance, external and internal examination of pups at necropsy were also recorded. In addition, thyroid hormone levels were determined in 1 pup/sex/group randomly selected at Day 14 post partum. Histopathological evaluation of reproductive organs/tissues was performed on all control and high dose males and females, as well as on all abnormalities detected during post mortem observation. The identification of the stages of the spermatogenic cycle was performed in the males from all dose groups.

Three study animals (1 male and 1 female dosed at 150mg/kg/day, 1 female dosed at 100mg/kg/day) were found dead or humanely sacrificed during the study. A number of clinical signs and post mortem macroscopic and microscopic changes were observed in these animals. Minor clinical signs (hunched posture, pallor) were detected in the high dose males and females from the third week in vivo phase (mating phase). These were not considered of toxicological relevance. Reductions in body weight/body weight gain or body weight losses were observed in males and females dosed at 100 and 150mg/kg/day, starting from the mating phase up to termination. Reductions in food consumption were observed in the females dosed at 150mg/kg/day, starting from the pre-mating phase up to termination. Two females dosed at 150mg/kg/day and 1 female dosed at 50mg/kg/day were found not pregnant, while 2 further females dosed at 150mg/kg/day showed total litter loss on Days 0 and 1 post partum. Increase in pre-coital interval, reduced copulatory indices (80% and 55.6% in the males dosed at 100 and 150mg/kg/day and 88.9% in the females dosed at 150mg/kg/day) and fertility indices (55.6% and 88.9% in males and females dosed at 150mg/kg/day, respectively) were also observed, with incidence and severity increasing with the dose. All the above findings are regarded as an effect of the test item on mating performance and fertility, mainly in the males. Effects on conception, development of conceptus and parturition were demonstrated by reductions in corpora lutea, implantation sites and total litter size in the females dosed at 150mg/kg/day, dose-related increases in the mean pre-natal loss (percentage) in all treated females and increased parturition length in the females dosed at 150mg/kg/day.

In addition, total litter size and live litter size, mean litter weights and/or mean pup weights were significantly reduced at birth, on Days 1, 4, 7 and/or 14 post partum in the females dosed at 100 and/or 150mg/kg/day, revealing an effect of the test item on the viability and growth of the offspring during the post partum period. No significant differences were observed in sex ratio at birth and on Days 4 and 14 post partum. All the above changes showed a dose-related trend, generally statistically significant. No nipples were seen and no significant differences were observed in the anogenital distance and weight of thyroid in treated pups, when compared to controls.

Some clinical signs and mortalities were observed in the pups of all treated groups, with severity and incidence increasing with the dose level. Necropsy findings in pups did not reveal any treatment-related effect. Thyroid hormones (T3, T4 and TSH) did not show any changes of toxicological relevance either in pups or in parental males. Post mortem observations of the parental animals revealed a reduction of terminal body weight (in males and females dosed at 150mg/kg/day), increases of the absolute and relative spleen weight (dosages of 100 and 150mg/kg/day), reductions of the absolute and/or relative thymus weight in both sexes (dosage of 150mg/kg/day) and absolute and relative epididymes weight of males dosed at 150mg/kg/day.

Swollen or enlarged spleen and reduced size of the thymus were seen in animals dosed at 150mg/kg/day, as well as instances of small epididymides, seminal vesicle and testes in the males of this group. Microscopic examination revealed treatment-related changes in the spleen (increase of yellow/brown pigmentation, congestion, lymphoid depletion and for high dose females extramedullary haematopoesis) of males and females dosed at 100 and 150mg/kg/day, in the thymus (atrophy) of males and females dosed at 150mg/kg/day and in the testes and epididymes (germ cell degeneration) of males of the same group. Seminiferous tubules showed regular layering in the germinal epithelium in all control and males treated at 50 and 100mg/kg/day.

In conclusion, effects on mating performance and fertility were demonstrated at 100 and 150mg/kg/day by the increase in pre-coital interval, reduced copulatory indices and reduced number of pregnant females. Effects on conception, development of conceptus and parturition were also demonstrated by reductions in corpora lutea, implantation sites, total litter size and increases in the mean prenatal loss (percentage), parturition length in the females and litters dosed at 150mg/kg/day. These signs were associated with reductions of total litter size, live litter size, mean litter weights and/or mean pup weights from birth to Day 14 post partum in the females and litters dosed at 100 and/or 150mg/kg/day, revealing an effect of the test item on the viability and growth of the offspring during the post partum period. These effects were associated to slight in vivo effects on body weight and food consumption and post mortem effects on the spleen and thymus of males and females dosed at 100 and 150mg/kg/day, which contributed to the observed effects on reproduction and development. The reduced fertility observed in the males dosed at 150mg/kg/day was associated to germ cell degeneration in the testes and epididymides seen in the males of this group. No adverse effects for general toxicity, reproductive and developmental toxicity were observed in male and female animals dosed at 50mg/kg/day.

The NOAEL (No Observed Adverse Effect Level) for parental toxicity, fertility and reproduction parameters was considered to be 50 mg/kg/day for males and females.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 June 2017 - 19 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ordered from Envigo RMS srl, San Pietro al Natisone (UD), Italy and obtatined by Envigo Netherlands, Kreuzelweg 53, 5961 NM Horst, Netherlands.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: ca 9-10 weeks
- Weight at study initiation: ca 275 g for males and ca. 200 g for females
- Fasting period before study: no
- Housing: up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages
- Diet (e.g. ad libitum): rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy)
- Water (e.g. ad libitum): tap water
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

The required amount of DIMERCAPTO-1,8-DIOXA-3,6-OCTANE (DMDO) was suspended in the vehicle. The formulations were prepared daily (concentrations of 5, 10 and 15mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
The test item was administered orally by gavage at a dose volume of 10mL/kg body weight.

Details on mating procedure:

Each group comprised 10 male and 10 female rats. Females were selected on the basis of pre-exposure oestrous cyclicity and animals that failed to exhibit regular cycles were not included in the groups.
Pairing was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (spermidentification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred or 14 days had elapsed. The pairing combination of 5 animals (2 dosed at 100mg/kg/day and 3 dosed at 150mg/kg/day) which did not have positive identification of mating after 14 days of pairing was changed within the treatment group (female nos. 51, 55, 63, 67, 69).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in RTC Study no. A2370 in the range from 1 to 20mg/mL. Linearity, accuracy and precision were within the limits stated in RTC SOPs for suspensions (r > 0.98; accuracy 90-110%; precision CV < 5%).
A 28 hour stability at room temperature and an 8 day stability at +5°C ± 3°C were verified in the range from 1 to 20mg/mL. The proposed formulation procedure for the test item was checked in the range from 1 to 20mg/mL by chemical analysis (concentration and homogeneity) in RTC Study no. A2370 to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in RTC SOPs for concentration (85-115%) and homogeneity (CV < 10%). Samples of the formulations prepared onWeeks 1 and 7 were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in RTC SOPs for suspensions (85-115% for concentration and CV<10% for homogeneity).

Duration of treatment / exposure:

Males for 52 days.
Females for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice (for a total of 49 to 69 days).

Frequency of treatment:

Once daily

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval (approximately 1 - 1.5 hour post-dose) was selected taking into consideration the presence of post-dose reactions. All observations were recorded for individual animals.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20.Dams were also weighed on Days 1, 4, 7 and 13 post partum and on the day of necropsy.

FOOD CONSUMPTION:
- The weight of food consumed by each cage ofmales and females was recorded weekly during the pre-mating period, starting from allocation. Individual food consumption for the females was measured on post coitum Days 7, 14 and 20 starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.

WATER CONSUMPTION: No

OTHER:
Thyroid hormone determination (T3, T4 and TSH)
Blood samples (approximately 0.8 mL) were taken as follows:
– Four surviving parental female animals samples were taken as part of the necropsy procedure.
– Four surviving parental male animals samples were taken under isolfuorane anaesthesia from the retro-orbital sinus.
Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by a multiplex assay, using LuminexMagpix system and the MILLIPLEX MAP Rat ThyroidMagnetic Bead Panel kit (Merk Millipore, cat. no. RTHYMAG-30K).
The determination was restricted to samples from all parental males from all main groups.

Oestrous cyclicity (parental animals):

Females were evaluated for oestrous cyclicity 7 days before allocation to groups and during the allocation phase. Animals that exhibited irregular cycle were not included in the study. Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was made for females assigned to groups. Pre-test vaginal smears data are not tabulated in the report, but were preserved with other raw data of the study. The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Vaginal smears were also taken on the day of necropsy.

Sperm parameters (parental animals):

The testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was initially performed in all animals in the control and high dose groups
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages.
The examination was then extended to include the testes and epididymes and the spermatogenic staging of the low (50 mg/kg/day - Group 2) and intermediate (100 mg/kg/day - Group 3) dose group animals.

Litter observations:

A parturition check was performed from Day 20 to Day 25 post coitum. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth when the parturition was defined complete (Day 0 post partum).
As soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1, 4, 7 and 13 post partum.
Pups killed or dying during the lactation periodwereweighed before the despatch to necropsy.
Observations were performed once daily for all litters.
All pups (surviving to treatment and not selected for culling) were sacrificed with the dams on Day 14 post partum.

Adjustment of litter size (culling) and pups selection for blood collection at necropsy
Culling - Day 4 post partum
On Day 4 post partum, all pups were weighed and litters in excess of 8 offspring were culled to 8 (4 males and 4 females, where possible) by a random selection.
For litters with less than 8 pups, two of the pups of the litter were sacrificed for serum hormone assessments. In this case preference was given to remove two female pups in order to retain more male pups for possible nipple retention on Day 14 post partum.

Nipple count at Day 13 post partum
The number of nipples/areolae in male pups were counted in each pup on Day 13 post partum (no nipples were observed, therefore these data were not reported).

Anogenital distance (AGD)
The AGD of each pup was measured on Day 1 post partum. The AGD was normalized to the cube root of body weight collected on Day 1 post partum.

Thyroid hormone determination (T3, T4 and TSH)
On Day 4, as a part of the necropsy procedure, blood samples (approximately 0.2 mL) were taken from 2 culled pups (1 male and 1 female, where possible). Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac puncture). Pooled samples from different pups of the same sex were performed, when necessary.
On Day 14 post partum, blood samples (approximately 0.5 mL) were taken from 2 pups (1 male and 1 female, where possible). Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac puncture). Pooled samples from different pups of the same sex were performed, when necessary.
Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by a multiplex assay, using LuminexMagpix system and the MILLIPLEX MAP Rat ThyroidMagnetic Bead Panel kit (Merk Millipore, cat. no. RTHYMAG-30K).
The determination was restricted to samples from pups on Day 14 post partum

Postmortem examinations (parental animals):

Euthanasia
Parental males and parental females sacrificed for humane reasons or for total litter loss were killed with carbon dioxide.
Parental females were killed by exsanguination under isofluorane anaesthesia.

Parental males
The males were killed after the mating of all females, after 52 days of treatment.

Parental females
The surviving females with live pups were killed on Day 14 post partum.

Necropsy
The clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed (excluding the early deceased animals) and the required tissue samples preserved in fixative and processed for histopathological examination.

Females
All females were examined also for the following:
– external and internal abnormalities;
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals)

Organ weights

From all animals completing the scheduled test period, the organs indicated below were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.
The organs were:
Adrenal glands, Brain, Epididymides, Kidneys, Liver, Ovaries with oviducts, Parathyroid glands (weighed and preserved with thyroid gland), Prostate gland, Seminal vesicles with coagulating glands, Spleen, Testes, Thymus (where present), Thyroid, Uterus – cervix

Tissue processing
Samples of all the tissues listed in section 4.5.6 were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).
The tissues required for histopathological examination were: Abnormalities, Adrenal glands, Brain (cerebrum, cerebellum, medulla/pons), Clitoral gland Epididymides, Kidneys, Liver, Mammary glands (males), Mammary glands (females), Ovaries with oviducts, Parathyroid glands, Pituitary gland, Penis,
Prostate gland (dorsolateral and ventral), Sciatic nerve, Seminal vesicles with coagulating glands, Spleen, Testes, Thymus (where present), Thyroid, Uterus – cervix, Vagina
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological
evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was initially performed in all animals in the control and high dose groups dying during treatment period or killed at the end of the study.
The examination was restricted as detailed below:
i Tissues specified above in all males and females from the control and high
dose groups killed at term.
ii Spleen and thymus from all males and all females of the low and mid-dose groups
killed at termination.
iii Tissues specified above from all animals killed or dying during the treatment
period.
iv All abnormalities in all groups.
The examination was then extended to include the testes and epididymes and the spermatogenic staging of the low (50 mg/kg/day - Group 2) and intermediate (100mg/kg/day - Group 3) dose group animals.

Postmortem examinations (offspring):

Pups that had completed the scheduled test period (Day 4 post partum orDay 14 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal.
All pups found dead in the cage were examined for external and internal abnormalities.
All culled pups sacrificed at Day 4 post partum were subjected to an external examination.
Sex was determined by internal gonads inspection.
All live pups sacrificed at Day 14 post partum were killed and examined for external abnormalities and sex confirmation by gonads inspection.
All pups with abnormalities were retained in an appropriate fixative.

Thyroid was weighed from one male and one female pup selected for blood collection of hormones determination and preserved in 10% neutral buffered formalin for possible histopathological examination. The thyroid weight was determined after fixation.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables, the significance of the differences amongst group means were assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n is more than 5. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of theWilliams test. The criterion for statistical significance was p<0.05.

Reproductive indices:

Group mean values were calculated for all parameters. Data from females which did not mate, with total resorption or non-pregnant and from dams without live young were excluded from group mean calculations as considered appropriate by the Study Director.
The following reproductive indices were calculated:

Males

Copulation Index (%) = no.of males mated ×100/ no.of animals paired

Fe r t i l i t y Index (%) = no.of males which induced pregnancy ×100 / no.of males paired

Females

Copulation Index (%) = no.of females mated ×100/ no.of females paired

Fe r t i l i t y Index (%) = no.of pregnant females ×100 / no.of females paired

Males and females
Copul atory Interval =Mean number of days between pairing and mating

Offspring viability indices:

Pre-birth loss was calculated as a percentage from the formula:

(no.of visible implantations - total litter size at birth) x 100/ no.of visible implantations

Pre-implantation loss was calculated as a percentage from the formula:

(no.of corpora lutea - no.of visible implantations ×100) /no.of corpora lutea

Pup loss at birth was calculated as a percentage from the formula:

(Total litter size - live litter size) x 100/ Total litter size

Cumulative pup loss on Day 4 post partum (before culling) was calculated as a percentage from the formula:

[Total litter size - live litter size at Day 4(before culling)] ×100/ Total litter size at birth

Post-natal loss on Day 4 post partum, after culling, was calculated as a percentage from the formula:

[Li v e l i t t e r s i z e on Day 4(a f t e r cul l ing ) -l i v e l i t t e r s i z e on Day 1] ×100/Li v e l i t t e r s i z e on Day 4(a f t e r cul l ing )

[Li v e l i t t e r s i z e on Day 4 (a f t e r cul l ing )-l i v e l i t t e r s i z e on Day 13] ×100/ Li v e l i t t e r s i z e on Day 4(a f t e r cul l ing )

Sex ratios were calculated at birth and on Days 4 and 14 post partum and were presented as the percentage of males per litter.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Table 2.
Hunched posture was observed in 1 male dosed at 150mg/kg/day on Day 11 of pre-mating phase, while pallor was observed in a single female of the same group on Day 1 post partum. Due to their episodical nature they were not considered of toxicological relevance.

Mortality:

mortality observed, treatment-related

Description (incidence):

Table 1.
A total of three study animals (nos. X0560070 male and X0560075 female, dosed at 150mg/kg/day and no. X0560053 female, dosed at 100mg/kg/day) were found dead or humanely sacrificed during the study.
Animal no. X0560070 - Found dead on Day 29 of mating phase. Decreased activity, hunched posture, piloerection and semi-closed eyes were seen prior to death.
Animal no. X0560075 - Found dead on Day 12 post coitum. Lethargy, cold to touch, staining at peri-genital region, dyspnoea, semi-closed eyes were the clinical signs observed in this animal prior to death. This animal was not pregnant.
Animal no. X0560053 - Sacrificed on Day 23 post coitum for difficulty in parturition. Piloerection, pallor and difficulty in parturition were the clinical signs observed in this animal prior to humane sacrifice on Day 23 post coitum.

For the mid- and high dose females, the most relevant findings observed at macroscopic observations were swollen or dark colour of spleen; in addition for the high dose female small thymus was seen. At microscopic observations, the most relevant findings were mild or moderate congestion associated with increased yellow/brown pigmentation in red pulp associated or not with lymphoid depletion and severe atrophy of thymus.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Tables 3 & 4.
A reductions in body weights, generally statistically significant, were seen in the males dosed at 150mg/kg/day from pairing to sacrifice and in the females of the same dose group during post coitum period, when compared to controls. Reduced body weight gain/body weight losses were also observed in the males dosed at 150mg/kg/day during pairing up to sacrifice and in the pregnant females dosed at 100 and 150mg/kg/day during the gestation period. These reductions were still evident on Day 4 post partum and disappeared from Day 7 post partum.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Table 5.
No effects on food consumption were observed in the males during the pre-mating phase of the study.
Reductions were seen in the females dosed at 100 and 150mg/kg/day on Day 8 of pre-mating phase and during the post coitum and post partum periods.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Thyroid hormone determination (Table 6)
No relevant differences were recorded. The statistically significant difference of triiodo- thyronine (T3) recorded between control and parental males dosed at 100 mg/kg/day was not dose-related, therefore considered unrelated to treatment.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Table 19.
Treatment-related changes were seen in the spleen of mid- (100 mg/kg/day) and high (150 mg/kg/day) dose males and females, in the thymus of males and females dosed at 150 mg/kg/day and in the testes and epididymides of males dosed at 150 mg/kg/day.
Males and/or females dosed at 150 mg/kg/day showed the following lesions:
– Spleen: in the red pulp area, minimal to moderate increase of yellow/brown pigment- ation, congestion, lymphoid depletion localized in the marginal zone/PALS in the white pulp area. In addition, high dose females showed extramedullary haemato- poesis (EMH), which was also observed in the liver of 4 out of 9 females treated at 150 mg/kg/day.
– Thymus: minimal to moderate atrophy in males, increased incidence and severity in females. The remaining treated males and females showed a comparable incidence to their concurrent controls.
– Testes: germ cell degeneration was only noted in 4 out of 9 rats from minimal to mild degree. This degenerative change included a number of degenerative features, such as tubular vacuolation, partial depletion of germ cells, degenerating (multinucleated or apoptotic) germ cells, and disordered arrangement of the germ cell layers, which influenced the seminiferous tubules in the first half of the cycle (stages I-V).
The germ cell degeneration of testes was also associated with luminal germ cell degeneration in the ducts of epididymides. The change was characterized by the presence of intact cells (often recognizable as round spermatids or spermatocytes) and/or cell debris intermixed with mature sperm within the ductular lumen and provides a sensitive indicator of testicular toxicity.

The mid-dose males and females only showed minimal to mild increase of yellow/brown pigmentation and congestion in males and females spleen, whereas minimal lymphoid depletion was noted in few instances only in female spleen.
The remaining sporadic lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted in all control and treated males dosed at 50 and 100 mg/kg/day.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Table 8.
No relevant differences were recorded on the oestrous cycle.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The mean pre-coital interval was increased in the females dosed at 100 and 150mg/kg/day, when compared to controls.
The number of copulation plugs were similar between control and treated groups.

Males (Table 9)
The copulatory indices were 100% for controls and animals dosed at 50mg/kg/day, 80% and 55.6% in animals dosed at 100 and 150mg/kg/day, respectively.
The fertility indices were 100% for controls, 90%, 100% and 55.6% in animals dosed at 50, 100 and 150mg/kg/day, respectively.

Females (Tables 9, 10, 11)
The copulatory indices were 100% in the controls and in females dosed at 50 and 100mg/kg/day, 88.9% in those dosed at 150mg/kg/day.
The fertility indices were 100% for controls, 90%, 100% and 88.9% in animals dosed at 50, 100 and 150mg/kg/day, respectively.

Two females dosed at 150mg/kg/day (Group 4, nos. X0560075 and X0560077) were found not pregnant (did not mate), while 2 further females of the same group (Group 4, nos. X0560069 and X0560073) showed total litter loss on Days 0 and 1 post partum, respectively. One female dosed at 100mg/kg/day (Group 3, no. X0560053) was humanely killed for difficulty in parturition on Day 23 post coitum, while 1 female dosed at 50mg/kg/day was found not pregnant (Group 2, no. X0560037).
The number of females with live pups on Day 13 post partum was: 10 in the control group, 9 in the groups dosed at 50 and 100mg/kg/day and 6 in the group receiving 150mg/kg/day.

Mean gestation period was statistically significantly increased in the females dosed at 150 mg/kg/day (23 days), when compared to the other groups (22 days). In this group, the gestation duration was between 22 and 23 days for all dams, excepted for two dams dosed at 150 mg/kg/day (X0560069 and X0560073) which showed 25 and 24 days of gestation, respectively.
Corpora lutea, implantation sites and total litter size at birth showed also statistically significant reductions in the females dosed at 150 mg/kg/day (-27%, -29% and -40%, respectively). No significant increases of pre-implantation loss were seen, while the mean pre-natal loss (percentage) was 2, 2.5 and 4.7-fold higher of control value in the females dosed at 50, 100 and 150 mg/kg/day, respectively.
These data suggest a treatment-related effect of the test item on female reproductive performance, in terms of conception and parturition.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive performance

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

spleen

thymus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

germ cells

seminiferous tubules

testes

other: Epididymides

Treatment related:

yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Cold to touch, pale aspect, apparently no food intake (milk) and small appearance associated with an increasing incidence of mortality, were the main clinical signs noted in pups from groups treated at 100 and 150 mg/kg/day. Some of these signs (pallor, small appearance, cold to touch) were also observed in individual pups from control and group dosed at 50 mg/kg/day with significant lower incidence.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Cold to touch, pale aspect, apparently no food intake (milk) and small appearance associated with an increasing incidence of mortality, were the main clinical signs noted in pups from groups treated at 100 and 150 mg/kg/day. Some of these signs (pallor, small appearance, cold to touch) were also observed in individual pups from control and group dosed at 50 mg/kg/day with significant lower incidence.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Litter data - Day 0 to Day 4 post partum (Table 11)
Reductions, generally statistically significant, of total litter size and live litter size (and consequently of mean litter weights) were observed at birth, on Days 1 and 4 post partum in the females dosed at 100 and/or 150 mg/kg/day, when compared to controls. The mean pup weights were also significantly reduced (-14%) in the females dosed at 150 mg/kg/day on Day 1 post partum. In addition, percentage of pup loss in females dosed at 150 mg/kg/day was 3.5-fold greater on Day 4 post partum than on Day 0.

- Litter data - Day 7 to Day 13 (Table 12)
No further reductions in live litter size or increased pup loss percentage were seen on Days 7 and 13 in females dosed at 100 and 150 mg/kg/day. Mean litter weight was still statistically significantly reduced on Day 7 post partum in females dosed at 150 mg/kg/day (-35%) and on Day 13 post partum in females dosed at 100 and 150 mg/kg/day (-17% and -38%, respect- ively). Mean pup weights still showed statistically significant reductions (-22% and -27%, respectively) at 150 mg/kg/day on Days 7 and 14 post partum.
The above findings indicate a dose-related effect of the test item on the size of litters at birth, on their viability and/or growth during the post partum period.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Tables 7
No relevant differences were recorded at thyroid hormones determination in male and female pups performed on Day 14 post partum.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant differences were observed at statistical analysis in the weight of thyroid in treated pups, when compared to controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 13 post partum did not reveal any treatment-related effect.

Other effects:

no effects observed

Description (incidence and severity):

- Sex ratio (Table 13)
No significant differences were observed in sex ratio at birth and on Day 4 and 14 post partum.

- Anogenital distance (Table 14) and nipple count
Slight statistically significant increases in the anogenital distance (normalised value), performed on Day 1 post partum, were seen between control and male and female pups from dams treated at 50 and 150 mg/kg/day. Due to the absence of dose relation, this finding was considered to be unrelated to treatment.
No nipples were observed in male pups.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

Critical effects observed:

not specified

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

In conclusion, effects on mating performance and fertility were demonstrated at 100 and 150 mg/kg/day by the increase in pre-coital interval, reduced copulatory indices and reduced number of pregnant females. Effects on conception, development of conceptus and parturition were also demonstrated by reductions in corpora lutea, implantation sites, total litter size and increases in the mean pre- natal loss (percentage), parturition length in the females and litters dosed at 150 mg/kg/day. These signs were associated with reductions of total litter size, live litter size, mean litter weights and/or mean pup weights from birth to Day 14 post partum in the females and litters dosed at 100 and/or 150 mg/kg/day, revealing an effect of the test item on the viability and growth of the offspring during the post partum period. These effects were associated to slight in vivo effects on body weight and food consumption and post mortem effects on the spleen and thymus of males and females dosed at 100 and 150 mg/kg/day, which contributed to the observed effects on reproduction and development. The reduced fertility observed in the males dosed at 150 mg/kg/day was associated to germ cell degeneration in the testes and epididymides seen in the males of this group. No adverse effects for general toxicity, reproductive and developmental toxicity were observed in male and female animals dosed at 50 mg/kg/day.

Based on the results of the present study, the test item may be considered toxic for reproduction and development at dose levels of 100 and 150mg/kg body weight/day. The NOAEL (No Observed Adverse Effect Level) for parental toxicity, fertility and reproduction parameters was considered to be 50mg/kg/day for males and females.

Executive summary:

The effects of the test item, DIMERCAPTO-1,8-DIOXA-3,6-OCTANE (DMDO), on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of conceptus, parturition and early lactation of the offspring were investigated, when administered orally to male and female Sprague Dawley SD rats. Groups of 10 males and 10 females received the test item by gavage at dosages of 50, 100 and 150mg/kg/day. The following investigations were performed on parental animals of all groups: mortality check, clinical signs, body weight, body weight gain, food consumption and mating per- formance, oestrous cycle evaluation for parental females (2 weeks before dosing, during pre-mating and mating phases, prior to necropsy), thyroid hormone determination only for parental males, litter data, macroscopic observations, organ weights and histopathological examination. Clinical signs, anogenital distance, external and internal examination of pups at necropsy were also recorded. In addition, thyroid hormone levels were determined in 1 pup/sex/group randomly selected at Day 14 post partum. Histopathological evaluation of reproductive organs/tissues was performed on all control and high dose males and females, as well as on all abnormalities detected during post mortem observation. The identification of the stages of the spermatogenic cycle was performed in the males from all dose groups.

Three study animals (1 male and 1 female dosed at 150mg/kg/day, 1 female dosed at 100mg/kg/day) were found dead or humanely sacrificed during the study. A number of clinical signs and post mortem macroscopic and microscopic changes were observed in these animals. Minor clinical signs (hunched posture, pallor) were detected in the high dose males and females from the third week in vivo phase (mating phase). These were not considered of toxicological relevance. Reductions in body weight/body weight gain or body weight losses were observed in males and females dosed at 100 and 150mg/kg/day, starting from the mating phase up to termination. Reductions in food consumption were observed in the females dosed at 150mg/kg/day, starting from the pre-mating phase up to termination. Two females dosed at 150mg/kg/day and 1 female dosed at 50mg/kg/day were found not pregnant, while 2 further females dosed at 150mg/kg/day showed total litter loss on Days 0 and 1 post partum. Increase in pre-coital interval, reduced copulatory indices (80% and 55.6% in the males dosed at 100 and 150mg/kg/day and 88.9% in the females dosed at 150mg/kg/day) and fertility indices (55.6% and 88.9% in males and females dosed at 150mg/kg/day, respectively) were also observed, with incidence and severity increasing with the dose. All the above findings are regarded as an effect of the test item on mating performance and fertility, mainly in the males. Effects on conception, development of conceptus and parturition were demonstrated by reductions in corpora lutea, implantation sites and total litter size in the females dosed at 150mg/kg/day, dose-related increases in the mean pre-natal loss (percentage) in all treated females and increased parturition length in the females dosed at 150mg/kg/day.

In addition, total litter size and live litter size, mean litter weights and/or mean pup weights were significantly reduced at birth, on Days 1, 4, 7 and/or 14 post partum in the females dosed at 100 and/or 150mg/kg/day, revealing an effect of the test item on the viability and growth of the offspring during the post partum period. No significant differences were observed in sex ratio at birth and on Days 4 and 14 post partum. All the above changes showed a dose-related trend, generally statistically significant. No nipples were seen and no significant differences were observed in the anogenital distance and weight of thyroid in treated pups, when compared to controls.

Some clinical signs and mortalities were observed in the pups of all treated groups, with severity and incidence increasing with the dose level. Necropsy findings in pups did not reveal any treatment-related effect. Thyroid hormones (T3, T4 and TSH) did not show any changes of toxicological relevance either in pups or in parental males. Post mortem observations of the parental animals revealed a reduction of terminal body weight (in males and females dosed at 150mg/kg/day), increases of the absolute and relative spleen weight (dosages of 100 and 150mg/kg/day), reductions of the absolute and/or relative thymus weight in both sexes (dosage of 150mg/kg/day) and absolute and relative epididymes weight of males dosed at 150mg/kg/day.

Swollen or enlarged spleen and reduced size of the thymus were seen in animals dosed at 150mg/kg/day, as well as instances of small epididymides, seminal vesicle and testes in the males of this group. Microscopic examination revealed treatment-related changes in the spleen (increase of yellow/brown pigmentation, congestion, lymphoid depletion and for high dose females extramedullary haematopoesis) of males and females dosed at 100 and 150mg/kg/day, in the thymus (atrophy) of males and females dosed at 150mg/kg/day and in the testes and epididymes (germ cell degeneration) of males of the same group. Seminiferous tubules showed regular layering in the germinal epithelium in all control and males treated at 50 and 100mg/kg/day.

In conclusion, effects on mating performance and fertility were demonstrated at 100 and 150mg/kg/day by the increase in pre-coital interval, reduced copulatory indices and reduced number of pregnant females. Effects on conception, development of conceptus and parturition were also demonstrated by reductions in corpora lutea, implantation sites, total litter size and increases in the mean prenatal loss (percentage), parturition length in the females and litters dosed at 150mg/kg/day. These signs were associated with reductions of total litter size, live litter size, mean litter weights and/or mean pup weights from birth to Day 14 post partum in the females and litters dosed at 100 and/or 150mg/kg/day, revealing an effect of the test item on the viability and growth of the offspring during the post partum period. These effects were associated to slight in vivo effects on body weight and food consumption and post mortem effects on the spleen and thymus of males and females dosed at 100 and 150mg/kg/day, which contributed to the observed effects on reproduction and development. The reduced fertility observed in the males dosed at 150mg/kg/day was associated to germ cell degeneration in the testes and epididymides seen in the males of this group. No adverse effects for general toxicity, reproductive and developmental toxicity were observed in male and female animals dosed at 50mg/kg/day.

The NOAEL (No Observed Adverse Effect Level) for parental toxicity, fertility and reproduction parameters was considered to be 50 mg/kg/day for males and females.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

key study

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

Effects on fetal and pup development and testicular toxicity were observed at dose-levels inducing mortality, well in excess of the maximal tolerated dose. Therefore, DMDO is not classified according to CLP criteria.