Dimethylbis(oleoyloxy)stannane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Read-across to DMTC (Dimethyltin dichloride CAS No. 753 -73 -1)

Data from the repeated dose studies on the dimethyltin dichloride.

Link to relevant study records

Reference

Reference 1

Endpoint:

fertility, other

Remarks:

developmental neurotoxicity study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

other: read across: target

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.6300 (Developmental Neurotoxicity Study)

Deviations:

not specified

Principles of method if other than guideline:

The primary objective of the current study was to examine and characterize the potential developmental neurotoxicity of DMT. The neurotoxicological effects of DMT exposure were characterized in a rat model using two separate dosing paradigms, the first to replicate the approach used earlier by Noland et al. and the second to use a more standard exposure paradigm as described in the US EPA Developmental Neurotoxicity Test Guidelines. In both studies, behavioural assays were conducted beginning at PND 11, and continued throughout development and into adulthood. Moreover, specific brain regions were analysed for apoptotic cell death and neuropathology.

E.A. Noland, D.H. Taylor, R.J. Bull, Monomethyl and trimethyltin compounds induce learning deficiencies in young rats, Neurobehav. Toxicol. Teratol. 4 (1982) 539–544.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

EXPERIMENT 1
One hundred and twenty nulliparous Sprague–Dawley (CD) female rats (Charles River, Raleigh, NC, USA), 53 days old, were housed in AAALAC-International accredited, temperature- and humidity-controlled rooms (16−21 °C and 40−70%, respectively) on Beta-Chip bedding with food (Purina Rodent Chow 5001) and filtered tap water ad libitum, and maintained on a reverse 12-h light:dark cycle (lights on at 1200). Females were housed in pairs until mating. Two weeks prior to DMT exposure, estrus cycles were monitored through daily vaginal smears. Females (30/dose) were then rearranged and paired according to synchronized estrus cycles (over 4 days).

EXPERIMENT 2
Eighty-seven (n=21 control, n=22 per DMT dose groups (3, 15, and 74 ppm) timed-pregnant Sprague–Dawley (CD) female rats (Charles River, Raleigh, NC, USA), were received at gestational day (GD) 2 (sperm-positive considered gestational day 0). Upon arrival, all females were housed individually as in Experiment 1, except for the light cycle (lights on at 0600) and the temperature (19–21 °C). The total experiment was replicated over ten cohorts of dams, with treatment counterbalanced across groups, such that births occurred two to four consecutive days each week.

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dimethyl tin dichloride was dissolved in distilled/deionized water at concentrations of 0, 3, 15 or 74 mg/l (0, 1.6, 8.1, or 40 ppm Sn). Rats received the DMT or water in polypropylene/polyethylene water bottles containing double ballbearing sipper tubes. A concentrated stock solution (740 mg/l) was prepared every two weeks and stored at −20 °C; dilutions were made from this stock. Water bottles were changed and weighed twice weekly, and rats were always weighed at the same time.

Details on mating procedure:

EXPERIMENT 1
After two weeks of DMT exposure, the females were bred by placing two receptive females (i.e. late-stage proestrus) with a breeder male near the end of the light cycle, and removed the next day at lights-on. Following this cohabitation period, females were individually housed, and maintained on the DMT solutions with food (Purina Formulab Chow 5008) throughout gestation and lactation. Assignment of treatment was counterbalanced across cohorts.

EXPERIMENT 2
Timed-pregnant animals were used.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted to verify the concentration and speciation of the methyl tins in the DMT solutions. The concentrated stock solution and solutions of the low and high test concentrations were sampled daily for 5 days from water bottles maintained under conditions of the animal exposure. Ion chromatography with inductively coupled plasma mass spectrometry was used to determine levels of MMT, DMT and TMT. Total tin levels were measured using inductively coupled plasma optical emission spectrometry. The limit of detection was 1 ng Sn/ml for DMT and TMT and 10 ng Sn/ml for MMT.

Total tin analysis revealed that the low concentration, as prepared, was about 10% higher than the nominal value, whereas the high concentration was only about 2% higher. The concentration of DMT did not decrease over days and neither MMT nor TMT were detected in the samples.

Duration of treatment / exposure:

Experiment 1:
Dosing was performed before mating (2 weeks), through gestation and lactation (PND21).

Experiment 2
DMTC exposure occured from gestational day 6 to day 21 of lactation.

Frequency of treatment:

daily

Details on study schedule:

Experiment 1:
Dosing was performed before mating (2 weeks), throughout gestation and lactation (PND21).

Experiment 2
DMTC exposure occurred from gestation day 6 to day 21 of lactation.

Dose / conc.:

0 ppm

Dose / conc.:

3 ppm

Dose / conc.:

15 ppm

Dose / conc.:

74 ppm

No. of animals per sex per dose:

Experiment 1: 30 females per dose.
Experiment 2: 22 females per dose group, 21 for control group.

Control animals:

yes

Details on study design:

EXPERIMENT 1
After two weeks of DMT exposure, the females were bred by placing two receptive females (i.e. late-stage proestrus) with a breeder male near the end of the light cycle, and removed the next day at lights-on. Following this cohabitation period, females were individually housed, and maintained on the DMT solutions with food (Purina Formulab Chow 5008) throughout gestation and lactation. Assignment of treatment was counterbalanced across cohorts.

The day of birth was designated postnatal day (PND) 0, and litters (n=13 control, 9 at 3, 74 ppm, and 10 at 15 ppm) were culled to 8 males on PND 1. In a few cases, female pups were kept to maintain equal litter sizes. On PND 21, remaining offspring were weaned and the littermates separated and housed individually on Beta-Chip bedding and provided food (Purina Rodent Chow 5001) and filtered tap water. Only male offspring, one from each litter, were tested in the different neurobehavioral tasks; each pup was evaluated in only one test. Offspring were weighed at least weekly throughout.

EXPERIMENT 2
Using the same concentrations as in Experiment 1, DMT exposure began at GD 6 and continued through gestation and lactation. Litters (n=21 control, n=22 at 3, 15 ppm, and n=20 at 74 ppm), were culled to 4 males and 4 females on PND 4. On PND 21, the offspring were weaned and the littermates separated and housed individually on Beta-Chip bedding and provided food (Purina Rodent Chow 5001) and filtered tap water ad libitum. Both male and female offspring (one from each litter) were tested in the different neurobehavioral tasks with the exception of the runway task, in which only males were tested.

Dams were weighed on the days that bottles were changed, and also on specific gestational and lactational days. All pups were weighed on specific postnatal days, and in addition, pups used for each of the neurobehavioral tests were weighed on the day of testing.

Parental animals: Observations and examinations:

Clinical signs, food consumption, water consumption, and body weight were recorded.

Litter observations:

RUNWAY LEARNING TEST
The runway learning test is an appetitive learning paradigm in which a food-deprived PND 11 rat pup was trained to negotiate a runway for a dry suckling reward from its anesthetized mother in the goal box. Briefly, the apparatus was a Plexiglas runway with a goal box at the end, which was maintained at 37 °C through the use of water-circulating heating pads. The pups learned to traverse the runway to reach the anesthetized dam and latency was recorded. Acquisition consisted of reinforced (R; 15 s of dry suckling) and non-reinforced (N; placement in a holding cage for 15 s) trials. If the pup failed to find the dam within the allotted time, the experimenter guided it down the runway for either (R) or to be immediately placed in the holding cage. Extinction (blocked access to the dam) immediately followed acquisition. The specific parameters for each experiment are outlined below.

Experiment 1:
Dams were anesthetized using 2 ml/kg i.p. Chloropent® equivalent. The dams were dosed approximately 15 min before testing began. Pups (n=11 control, 9 at 3 and 74 ppm, and 10 at 15 ppm) were food-deprived for 10 h prior to testing, and tested during their dark cycle. Acquisition consisted of 25 alternating (R) and (N) trials, with a maximum of 120 s for each trial and an inter-trial interval of 15 s in a holding cage. Extinction trials began on the 26th trial, and the maximum time was set at 100 s. When pups reached the criterion of two consecutive 100-second trials, they were no longer tested. All pups were tested until they met criterion, regardless of the number of trials required.

Experiment 2:
Dams were anesthetized using Nembutal sodium solution (0.65 ml i.p.; 50 mg/ml). A different training schedule was used in Experiment 2. In this paradigm, pups (n=20/dose except n=19 at 74 ppm) were food-deprived for 8 h and then tested during their light cycle. Testing began with a preliminary training session of 5 massed (R) trials, followed by a 2 min retention interval in the holding cage. There were then 25 acquisition trials in which (R) and (N) trials alternated in blocks of 5 trials, beginning and ending with 5(R) trials, with an 8-second inter-trial interval. The maximum time allowed for each trial was 100 s. Extinction trials began on the 26th trial, and the criterion to extinction was one trial with a 100-second latency. A maximum of 10 extinction trials were run.

MOTOR ACTIVITY
Motor activity data were collected using automated figure-eight chambers. Photocell interruptions (counts) were recorded over 5-min intervals of the 30-minute test session. In Experiment 1, motor activity was assessed in males at PNDs 13, 17 and 21 (n=14 control, n=9 at ppm, n=10 at 15 ppm, and n=9 at 74 ppm). In Experiment 2, only PND 17 male and female offspring (one male and one female from each litter; n=21 control and 15 ppm, n=20 at 3 ppm, and n=17, 74 ppm) were tested.

SPONTANEOUS ALTERATION (Experiment 2 only)
Spontaneous alternation was measured on PND 25 using a Plexiglass T-shaped apparatus. Although others have not reported adult levels of alternation (85–90%) until one month of age or older, pilot studies in our laboratory indicated that in our two-choice alternation task, using a freechoice, continuous exploration protocol, pups achieved 80% alternation by PND 25. Thus, in the present study, PND 25 pups were placed in the stem for a 30 s acclimation, after which time the gate was raised allowing the rat to enter either arm. The rat was then allowed to explore freely between only the two
opposing arms for 5 min. All arm entries were counted as the measure of motor activity, whereas alternation was considered when the rat left one arm and entered the other. A minimum of six arm entries was required to calculate alternations to avoid erroneous data due to low activity. Percent alternation was calculated as the number of opposite arm entries divided by the total arm entries less one (to subtract the first entry). Both males and females were tested at 10/dose.

MORRIS WATER MAZE (Experiments 1 and 2)
Spatial memory was evaluated using a Morris water maze. Rats were tested as adolescents/young adults (beginning about 7 weeks old in Experiment 1, 12 weeks old in Experiment 2). Each test trial was videotaped and the image digitized for computer analysis using maze-tracking software. Dependent variables included swim speed, latency and path length to find the platform, and time spent in the outer edge of the tank or one of the three concentric zones.

For spatial training, rats learned the fixed position of the platform during 2 trials a day with an inter-trial interval of 5 min, for 9 days. The starting position was semi-randomly varied (all four starting positions were used before one was repeated, but the order itself never repeated) every 2 days. The maximum trial time was 60 s, after which time the observer guided the rat to the platform. On the 10th day, a probe trial was conducted in which the platform was removed and the subject's tendency to search in the correct quadrant was measured over 60 s. Dependent variables were the Gallagher proximity score, and percent total time within each quadrant. A visible probe trial was also conducted using a raised platform of a contrasting colour to confirm that the tested animals were not visually impaired.

In Experiment 1, only males were tested in the water maze (n=11 control, 7 at 3 ppm, 9 at 15 ppm, and 11 at 74 ppm) whereas both males and females (one from each litter) were tested in Experiment 2 (n=10/sex/dose).

Postmortem examinations (offspring):

NEUROPATHOLOGY (Experiments 1 and 2)
Experiment 1:
For neuropathological evaluations, male rats (n=6–8/dose at PND1, n=5–8/dose at PND 12, n=5–9/dose at PND22, and n=5/dose at adult age, 80–90 days old) were deeply anesthetized with pentobarbital and perfused via the left ventricle with buffered 4% formaldehyde:0.1% gluteraldehyde. Sagittal blocks of tissue were embedded in paraffin and sectioned to include all major structural landmarks of the brain in each section (e.g., olfactory bulb, striatum, cerebral cortex, hippocampus, thalamus, hypothalamus, brainstem, cerebellum). Twenty-four sections of brain from each rat, from each age, at each dose, were stained with hematoxylin and eosin.

Brains from all control and high-dose rats at all ages were evaluated by a certified pathologist. Step-down assessments, i.e., evaluation of the lower dose groups, were only conducted in the adult rats due to the remarkable findings in the high-dose group. Scoring of the severity of observed changes was conducted with the pathologist blind to the dosage group.

Experiment 2:
Brains were prepared and examined as described for Experiment, but only adult rats (both males and females) were used (n=10/dose/sex, except n=9 15 ppm males).

BRAIN WEIGHTS (Experiments 1 and 2)
Male rats were decapitated under CO2-induced anesthesia at PND1, 12, 22, and as adults (Experiment 1; n=4–11/dose/age) or PND12, 22, and as adults (Experiment 2; n=7–9/dose/age). From PND12 on, all subjects came from different litters. Brains were quickly removed and weighed.

APOPTOSIS ASSESSMENT
Apoptosis was quantified using a Cell Death ELISA procedure. Modifications to the procedure in the kit have been described previously; the kit has been adapted for use with intact tissue and validated with both fresh and frozen brain tissue. Moreover, the results have been corroborated qualitatively by agarose gel and TUNEL data. In short, the enzyme-linked immunosorbent assay (ELISA) uses antibodies to bind fragmented DNA characteristic of apoptotic cell death. The bound fragments (i.e. nuclesomes) are then quantified photometrically.

Experiment 1:
After weighing, brains were dissected free-hand into the following regions: brainstem, neocortex, hippocampus and cerebellum. Each region was immediately frozen in 2-methyl butane on dry ice for 30 s and stored at −70 °C [30]. ELISA assays were conducted only on tissues collected at PND22 (n=3–7/dose/region) and as adults (n=2–4/dose/region).

Experiment 2:
Tissues were collected from male rats only as described for Experiment 1 on PND12, 22, and as adults, with n=6–8/dose/region.

Statistics:

Where data were collected from littermates, they were analysed using litter as the unit (e.g., sex or multiple observations nested within litter). Continuous data (e.g., body weight, water intake, activity counts, etc.) were analysed using a general linear model ANOVA. Extinction in the runway test was analysed with a survival model for censored data and count data (e.g., number of pups learning, pregnancy rate) were compared using Fisher's exact test. In the second runway study, within-subject trend analyses (SAS) of the slopes of each 5-trial block were used to evaluate whether the latencies were increasing or decreasing (significantly different from a slope of zero) over the 5 trials, or whether there was no change (slope of zero). Whenever the same rat was used in repeated tests (e.g., repeated motor activity testing, body weights over time, within-session activity), the analyses included time (or interval) as a repeated factor; however, if no interaction between time/interval and treatment was revealed, data were collapsed across time. Likewise, if there was a significant interaction between sex and dose (or sex, dose, and age/time), only then were males and females analysed separately to evaluate dose (and/or age/time) effects. Where sex interactions were not significant, the data were combined. Dunnett's t-test was used to compare dose groups with the control. In all cases, resulting twotailed probability values ≤0.05 were considered significant.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Experiment 1: Only the high concentration altered maternal weight gain, which was significantly lower than control throughout exposure.

Experiment 2: Reductions in body weight were not evident until lactation, at which time the high-concentration body weight was significantly lower than controls.

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Experiment 1: Overall, water consumption increased among all dose groups throughout gestation and lactation. In the first two weeks of exposure (premating), consumption was significantly decreased in all DMT-exposed groups. During the first half of gestation, only the high concentration decreased consumption. Although the high-concentration consumption appeared lower throughout the rest of exposure, these differences were not significantly different from control.

Experiment 2: From the beginning of exposure to the end of gestation, fluid intake was significantly lower in the 15 and 74 ppm dose groups. Additionally, the 3 ppm dose group showed decreased consumption during the second week of exposure. Intake returned to control levels in all but the high-concentration group during lactation.

Reproductive performance:

no effects observed

Description (incidence and severity):

Experiment 1: The overall pregnancy success rate was low (47 of 120 rats, 39%). There was no treatment effect on the number of pregnancies: n=10 control, 14 at 3 and 15 ppm, and 9 at 74 ppm. All births occurred within a 24-hour time frame and there was no effect of treatment on the number of pups per litter which supports the conclusion that maternal gestational integrity was unaffected by DMTC.

Experiment 2: All of the timed-pregnant females delivered with the exception of one 74 ppm DMT female. Additionally, one 74 ppm female delivered only six pups and was not used. All of the deliveries occurred when expected and there was no effect of treatment on the number of pups per litter which further supports the conclusion that maternal gestational integrity was unaffected by DMTC.

Dose descriptor:

LOAEL

Effect level:

74 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

water consumption and compound intake

Dose descriptor:

NOAEL

Effect level:

15 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

water consumption and compound intake

Key result

Dose descriptor:

NOAEL

Effect level:

74 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: Exp 1: No effect on number of pregnancies. All births occurred within 24-hour, with no effect on number of pups per litter. Exp 2: No effect of treatment on number of pups per litter. Maternal fertility and gestational integrity was unaffected by DMTC.

Critical effects observed:

not specified

Lowest effective dose / conc.:

74 ppm

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Experiment 1: The total numbers of live pups per litter were (mean ±SEM): control, 14.4±1.0; 3 ppm, 13.3±0.7; 15 ppm, 15.0±0.9; and 74 ppm, 12.8±0.8. Statistical analysis suggested a trend toward fewer males in the treated groups (dose F(3, 43)= 2.35), however the p value [p=0.085] was not less than 0.05, and there there was no significant treatment-by-sex interaction. The suggested trend may have been due to the three litters in the high-concentration group which had one to two dead pups, whereas control litters had none; however, there were no statistically significant differences in litter size across groups. There were no external anomalies reported in controls or in any treatment group. After culling at PND1, three litters (one control, two at 74 ppm) lost three to four more pups each, again not a difference of biological significance.

Experiment 2: The total number of live pups per litter was not significantly different across groups, thus diminishing the relevance of the suggested trend observed in Experiment 1. There were no external anomalies reported in controls or in any treatment group.

Conclusion: These data support the conclusion that DMTC was not embryotoxic in either experiment and that the gestational integrity of the conceptus was unaffected by DMTC at any dose.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Experiment 1: There were no statistically significant differences in (male) pup weights across groups at any postnatal period or when pups were weighed prior to each of the neurobehavioral tests. Female pups were maintained to preserve equal litter sizes across groups, but female pup weights were not reported. It is inferred that differences would have been reported had they occurred.

Experiment 2: Body weight changes among the pups during the lactation period showed a significant dose-by-sex interaction (F(3, 69)=3.0, p=0.037). Step-down analyses of males and females revealed that males in the high-concentration group weighed significantly less than controls throughout lactation, and the decrease in females in the same group reached significance only at PND 17 and 21. This effect was clearly transient. There were no treatment-related effects on body weight which was measured weekly after weaning, nor was there an effect at any time when offspring were weighed prior to neurobehavioral testing.
These data are equivocal and confounded by the experimental design. A statistical difference in body weights of the high-dose offspring was observed only after birth and only in Experiment 2, the segment with the shorter exposure period. This was not an in utero effect. The observed differences were transient and it cannot be determined with certainty whether this observation was a direct effect of postnatal exposure of the neonates in Experiment 2 or a result of poor maternal-neonatal interaction in the second experiment.

Conclusion: These data support the conclusion that DMTC was not fetotoxic in either experiment at any dose. In addition, an observed decrease in neonatal body weight of the high dose pups during lactation was equivocal and transient. Body weights for all treated groups of offspring recovered to normal after weaning.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Brain weight
Experiment 1: Analysis of brain weights revealed an overall effect of dose (F(3, 88)=3.61, p=0.016) but no interaction with age.
The data showed significant decreases in the low- and high dose groups when collapsed across the ages; overall, the low dose was 4% lower, and the high dose 8% lower, than controls. The mid dose group average was equal to the control mean.

Experiment 2: As in the first experiment, there was an overall effect of dose (F(3, 67)=4.05, p=0.01) in male rats but no interaction with age. Collapsed across age, only the high dose group showed a significant decrease of 4%.

Conclusion: Brain weights were recorded from animals at four different ages [PND1, 12, 22, and as adults] in Experiment 1. In Experiment 2 brain weights were recorded at three different ages [PND12, 22, and as adults]. The analysis collapses these into a single assessment of brain weight. These data support the conclusion that exposure to DMTC may adversely affect brain weight, however the effect cannot be clearly ascribed to a developmental effect because DMTC is known to be neurotoxic in adult animals and the observations in these experiments could be the result of postnatal exposure alone.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Apoptosis
Experiment 1: Significant dose-by-age interactions were observed for the cerebellar (dose-by-age F(3, 27)=2.93, p=0.05) and cortical (dose-by-age F(3, 27)=5.79, p=0.003) DNA fragmentation data, which were then determined to be significant decreases only at PND22 in the cerebellum (15 and 74 ppm) and cortex (all doses). These changes, however, did not show a clear dose–response. No differences in DNA fragmentation were observed in any of the brain regions for adult animals.

Experiment 2: A dose-by-age interaction (F(6, 60)=11.90, pb0.0001) for the brainstem data presented no treatment effect at PND12, with significant increases relative to control at PND22, and decreases in adults. These significant effects were seen in the mid and high dose groups. These changes may represent a shift in the normal decrease observed in control rats. In addition, cerebellar data revealed a small but significant increase (dose-by-age F(6, 60)=3.36, p=0.006) in DNA fragmentation at PND12, but only at the high dose (means ±SE: control, 1.0±0.07; 3 ppm, 1.30±0.07; 15 ppm, 1.06±0.04; 74 ppm, 1.07±0.08).

Conclusion: These data are inconsistent on three levels: location, direction and age. Brain apoptosis was recorded for four different brain regions [brainstem, neocortex, hippocampus and cerebellum] and from animals at two different ages [PND 22, and adult] in Experiment 1. In Experiment 2 brain apoptosis was recorded for the same four brain regions and from animals at three different ages [PND 12, 22, and adult]. The significant differences observed in Experiment 1 were decreases in the cerebellum and cortex, and only at PND22, not in adults. Whereas the significant differences observed in Experiment 2 were: in brainstem [PND22] where the difference was an increase; in cerebellum [PND12] where the difference was an increase; and in brainstem [adults] where the difference was a decrease. NO differences were observed in cortex.
The interpretation of these results is confounded by the normal decrease in apoptosis known to occur in control rats, a progression which was confirmed in this study. Overall, these data support the conclusion that exposure to DMTC may affect brain apoptosis, however the effect is inconsistent and cannot be clearly ascribed to a developmental effect because DMTC is known to be neurotoxic in adult animals and the observations in these experiments could be the result of postnatal exposure alone.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Neuropathology
Experiment 1: Histopathological alterations in the brain of offspring of dams exposed to DMT were noted in the cerebral cortex of rats sacrificed at PND22 and as adults. Three of five (60%) adult offspring at 74 ppm and one of five (20%) PND22 rats at 74 ppm had minimal/slight vacuolation of the neuropil of the gray matter of the cerebral cortex. Step-down evaluations of the lower dose groups showed similar vacuolation at 15 ppm (1 of 5 adults) and 3 ppm (1 of 5 adults). There were no lesions in the offspring at PND1 or 12, or in the offspring at the lower doses at PND22. The cerebral cortical lesion was characterized by 2–4 micron diameter, round vacuoles in the gray matter neuropil in the region of the orbital cortex. On a score of 1 (minimal) to 5 (severe), the rats in the lower dose groups received scores of 1, whereas the high-dose rats received scores of 2 (slight/mild).

Experiment 2: One male offspring at 74 ppm had a single neuron in the midbrain with central chromatolysis. The significance of this finding in a single neuron in a single treated rat remains undetermined.

Conclusion: The lack of neural histopathological anomalies in Experiment 2 suggest the results from Experiment 1 should be classified as equivocal.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

> Runway testing
Experiment 1: Using a criterion of having at least one latency less than the maximum time of 120 seconds, several pups in each treatment group failed to learn the task; however, there was no treatment-related difference in the incidence of non-learners (control, 3 of 12; 3 ppm, 1 of 9; 15 ppm, 0 of 10; and 74 ppm, 1 of 9). In the extinction phase, the median number of trials to reach the criterion (two consecutive trials of 100 seconds) for each dose group was: control, 18; 3 ppm, 13; 15 ppm, 15.5; and 74 ppm, 24. Again, there was no reported treatment-related difference in the outcomes.

Experiment 2: This segment used the massed-alternation paradigm and the same criterion as above for learning success of the runway task. The proportion of pups that failed to learn were control, 3 of 20; 3 ppm, 5 of 20; 15 ppm, 6 of 20; and 74 ppm, 6 of 19. There were no statistically significant differences in these outcomes. Analysis of the within-subject slopes (linear trend analysis) for each 5-trial block was much less variable. Learning, as defined by decreasing latencies during the reinforced blocks, gives a negative slope, whereas extinction, or increasing latencies (or no change in latency) during non-reinforced blocks, gives either a positive or zero slope. Such a pattern shows the contingency control over the behavior. Both the control and low-dose groups showed significant decreasing slopes (p'sb0.04 for all) for each of the set of R-trials, and the slopes during N-trials were not different from zero. In contrast, the 15-ppm group did not show decreased latencies on any of the R-trials, and the only significant slope change (p=0.006) was in the first N-trial (increasing latencies). The high-dose group showed a decreasing slope (p=0.008) only during the last block of R-trials. Thus, learning was not apparent during any R-trial blocks in the 15-ppm group, and was only achieved at the last set of trials in the 74-ppm group.
A number of pups did not extinguish the behavior in 10 trials, and therefore did not meet the criterion for extinction: control, 4 of 17; 3 ppm, 2 of 15; 15 ppm, 3 of 14; and 74 ppm, 1 of 13. Thus the expected outcome, extinction of the behavior, did not occur in 23.5%, 13.3%, 21.4%, and 7.7% of the pups for the control, low, mid and high dose groups, respectively. While this pattern of unlearning the task appears to suggest the inverse of the learning pattern, there were no statistically significant differences in these outcomes.

Conclusion: DMTC had no consistent statistically significant effect on the development of runway learning as a behavioral maturation parameter.

> Motor activity
Experiment 1: Total motor activity counts during 30 minute sessions increased with the age of the animals when tested on PND 13, 17 and 21, however there were no treatment-related differences in the activity levels. Analysis of the within-session activity (in 5-min intervals) showed that habituation (significant change in activity across intervals) was not evident until PND 21 in all treatment groups. This is not a treatment-related adverse outcome.

Experiment 2: Total counts for each group (sexes combined, mean ±SEM) were: control, 115.4±7.8; 3 ppm, 114.2±5.6; 15 ppm, 121.5±9.2; and 74 ppm, 119.0±8.8). Unlike in Experiment 1, habituation was evident in all treatment groups at PND 17, but this is not a treatment-related adverse outcome.

Conclusion: These data support the conclusion that DMTC had no effect on the development of motor activity as a behavioral maturation parameter.

> Spontaneous alternation (Experiment 2 only)
The percent of alternations showed no significant difference across groups or gender. It can be concluded that DMTC had no effect on the development of spontaneous alternation as a behavioral maturation parameter.

> Morris water maze
[This is an integrated behavioural assessment which includes assessment of maze learning and swim speed, and an evaluation of test animals’ search parameters]
Experiment 1: All groups eventually learned; however, the middle dose group showed significantly longer latencies during the first week of training (dose F(3, 31)=3.57, p=0.025). Analyses of the spatial distribution of swimming indicated that, in the second week, the low and middle dose groups spent significantly less time in the middle zone (dose F(3, 31)=5.79, p=0.003), and in addition, the 15 ppm group spent more time in the outer zone (dose F(3, 31)=5.52, p=0.004). This represented a less efficient search strategy, since the platform was located in the middle zone, and may have influenced the longer latencies observed in this treatment group. The high dose group showed no differences on any of these parameters.
In this segment swim speed was not affected either positively or negatively by treatment with DMTC at any dose. In this segment neither the memory probe search parameter [platform removed after its location is learned] nor the visual probe search parameter [platform raised above water surface to facilitate its acquisition as a target] were affected by treatment with DMTC at any dose.

Experiment 2: As in Experiment 1, the middle dose group had significantly higher latencies to learn the platform position (dose F(3, 71)=3.1, p=0.032). This was seen in both sexes in the second week, as evidenced by the lack of a treatment-by-sex interaction. There were, however, differences between the sexes in terms of the spatial pattern of swimming (first week, middle zone block-by-trial-by-dose by-sex F(12, 284)=2.03, p=0.022; outer zone block-by-trial by-dose-by-sex F(12, 284)=1.87, p=0.038). In the first week of training, females spent less time in the middle zone, and more time in the outer zone. On days 2 and 3, this pattern was significant in all dose groups. This propensity for the outer zone persisted in the middle dose group into the second week of training (dose F(3, 71)=7.44, p=0.0002), and was significant for both males and females. In both cases, the 15 ppm rats spent more time searching in the outer zone, and less time in the middle zone of the tank.
Analysis of the memory probe again revealed less time in the middle zone (dose F(3, 71)=3.21, p=0.028), in both males and females of the middle dose group. The time in the quadrants differed (dose-by-quadrant F(3, 71)=4.22, p=0.008), with the low dose group showing significantly less time in the correct quadrant, and the middle dose group showing a similar trend (mean % time in correct quadrant ± SE: control, 48.3±2.2%; 3 ppm, 39.7±3.0; 15 ppm, 40.6±2.2%; 74 ppm, 48.6±2.2%).
In this segment, as before, swim speed was not affected either positively or negatively by treatment with DMTC at any dose. The visual probe search parameter was not affected by treatment with DMTC at any dose. Outcomes for the memory probe search parameter were not reported.

Conclusion: These data support the conclusion that in utero and postnatal exposure to DMTC did not affect the development of integrated behavioral assessments which included assessment of maze learning and swim speed. The evaluation of test animals’ search parameters caused significantly higher latencies to learn the platform position in both sexes at the mid dose only. The lack of any significant differences at either a five-fold higher dose or at a dose one-fifth of that producing the outcome suggest the result may be anomalous and should be classified as equivocal. The lack of a dose-response in two separate and similar experiments supports the conclusion that in utero and postnatal exposure to DMTC did not consistently effect the development or maturation of the integrated neurobehavioral assessments or the test animals’ search capabilities.

Dose descriptor:

NOAEL

Generation:

F1

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable

Critical effects observed:

yes

Lowest effective dose / conc.:

15 ppm

System:

central nervous system

Treatment related:

yes

Dose response relationship:

yes

Reproductive effects observed:

no

Table 1: DMTC intake (mg/kg/day) calculated from average fluid consumption and body weights for each phase of Experiments 1 and 2

	Concentration (ppm)	Intake (ml/d)	Weight (g)	DMTC (mg/kg/d)
Experiment 1
Pre-mating	0	27.6	266.1	-
	3	24.1^a	255.0	0.28
	15	20.4^a	263.3	1.16
	74	14.7^a	248.6^a	4.38
Gestation	0	37.5	332.4	-
	3	34.7	321.0	0.32
	15	34.1	331.4	1.54
	74	29.6^b	307.2^a	7.13
Lactation	0	61.9	339.5^a	-
	3	58.0	336.2	0.52
	15	58.3	342.7	2.55
	74	50.3	304.2^a	12.2
Experiment 2
Gestation	0	36.8	301.0	-
	3	32.1^b	296.3	0.33
	15	30.1^a	295.8	1.53
	74	28.2^a	287.6	7.26
Lactation	0	62.8	324.3	-
	3	60.9	318.3	0.57
	15	56.7	318.3	2.67
	74	47.6^a	297.1^a	11.9

 

^a Indicates data significantly different from control throughout the period measured.

^b Indicates data significantly different from control during part of the period measured.

Conclusions:

The results support the following conclusions with respect to the effects of perinatal exposure to DMTC:
- maternal toxicity expressed as decreased maternal fluid consumption and depressed maternal weight gain at the high concentration (74 ppm) [in both DNT studies];
- no treatment-related embryotoxicity measured as gestational integrity [in both studies];
- decreased postnatal growth only during lactation with evidence of F1 recovery thereafter [Study 2 only];
- no significant effect on either of two memory probes [in both studies];
- altered the spatial learning in the Morris water maze, but only at the intermediate concentration (15 ppm) [no dose-response in either study];
- decreased F1 brain weight [consistent only at a maternally toxic dose (74 ppm) in both studies];
- inconsistent and minimal to slight histopathological alterations in F1 brain, characterized as vacuolation of the cortex [in Study 1 only at high dose on PND22 and all doses as adults, no findings in Study 2 at any dose];
- inconsistent altered levels of an apoptotic marker (DNA fragmentation) [in Study 1 decreased at PND22 at all doses but not in adults at any dose; in Study 2 no differences at PND12 at any dose, increased at PND22 at mid and high doses but decreased in adults at the same dose levels].
Comment: The age and brain area at which this was seen was not consistent and no effect was consistent across the two studies or at any dose.

Executive summary:

Two developmental neurotoxicity studies were conducted with dimethyltin dichloride administered in drinking water at 3, 15 and 75 ppm. In the first study, female Sprague–Dawley rats were exposed 2 weeks prior to mating and throughout mating, gestation and lactation. Male offspring were compared to controls for differences in: 1) pre-weaning learning in an associative runway task, 2) motor activity ontogeny, 3) spatial learning and retention in the Morris water maze as adults, 4) brain weight, 5) biochemical evidence of apoptosis, and 6) neuropathology. Maternal toxicity was expressed as decreased maternal weight gain and decreased fluid intake. Only in the first of the two studies was developmental toxicity expressed as minimal to slight brain histopathology vacuolization of F1 adults at all doses, decreased brain weights, and one of several neurobehavioral end points was altered.

In the second study, exposure to timed pregnant Sprague–Dawley rats occurred via drinking water from gestational day 6 to weaning and a similar battery of behavioral tests was administered to the F1 animals. The high concentration again depressed maternal weight gain and fluid intake. The F1 animals at the high dose had lower weight only during lactation, not thereafter. There was decreased brain weight but no correlating adverse histopathological findings in high-dose offspring. No significant differences in motor activity or in the spontaneous alteration activity test occurred as the result of treatment. Statistically significant learning deficits were not observed in the runway test, but a difference from controls was observed in mid-dose animals only in the water maze test.

The results of both studies demonstrate a lack of dose-response in spatial learning after perinatal DMT exposure. Changes in brain weight, and the occurrence of neuropathological lesions in offspring at the high dose indicate a potential for neurotoxicity of DMT at a maternally toxic dose. Histopathological alterations of minimal to slight severity in the brain of offspring of dams exposed to DMT in Experiment 1 were noted in the cerebral cortex of rats sacrificed at PND22 and as adults. These lesions were not observed in Experiment 2 in any brain region. The NOAEL was determined to be 15 ppm (equivalent to 1.16-2.67 mg/kg/day).

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across to structurally similar substance dimethyltin dichloride (DMTC) (EC Number 212-039-2, CAS Number 753-73-1), see attached justification.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

dose level:

Effect level:

74 mg/L drinking water

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/L drinking water

Based on:

test mat.

Sex:

female

Key result

Dose descriptor:

NOAEL

Effect level:

3 mg/L drinking water

Based on:

test mat.

Sex:

female

Critical effects observed:

not specified

Dose descriptor:

NOAEL

Generation:

F1

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable

Critical effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Read-across to DMTC (Dimethyltin dichloride CAS No. 753 -73 -1)

Available data indicate that fertilisation and implantation of fertilised ova were unaffected by dimethyltin chloride when compared to control rats. No effects, either in organ weights or at histopathology, were ever detected for any organ of the male and female reproductive system in the available repeated-dose toxicity studies. These data support the conclusion that the DMTC does not adversely affect fertility in rats.

Short description of key information:
Dimethyltin dichloride was maternally toxic and fetotoxic. Dimethyltin dichloride did not adversely affect fertility.

Effects on developmental toxicity

Description of key information

Read-across to DMTC (Dimethyltin dichloride CAS No. 753 -73 -1)

The NOAEL value from the Ehman (2007) paper was selected as the key value on a worst case basis.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

other: read across: target

Qualifier:

equivalent or similar to guideline

Guideline:

other: U.S. Environmental Protection Agency, Developmental Neurotoxicity Study, Health Effects Test Guidelines, OPPTS 870.6300, EPA 712-C-98- 239,

Deviations:

not specified

Principles of method if other than guideline:

The primary objective of the current study was to examine and characterize the potential developmental neurotoxicity of DMT. The neurotoxicological effects of DMT exposure were characterized in a rat model using two separate dosing paradigms, the first to replicate the approach used earlier by Noland et al. and the second to use a more standard exposure paradigm as described in the US EPA Developmental Neurotoxicity Test Guidelines. In both studies, behavioural assays were conducted beginning at PND 11, and continued throughout development and into adulthood. Moreover, specific brain regions were analysed for apoptotic cell death and neuropathology.

E.A. Noland, D.H. Taylor, R.J. Bull, Monomethyl and trimethyltin compounds induce learning deficiencies in young rats, Neurobehav. Toxicol. Teratol. 4 (1982) 539–544.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

EXPERIMENT 1
One hundred and twenty nulliparous Sprague–Dawley (CD) female rats (Charles River, Raleigh, NC, USA), 53 days old, were housed in AAALAC-International accredited, temperature- and humidity-controlled rooms (16−21 °C and 40−70%, respectively) on Beta-Chip bedding with food (Purina Rodent Chow 5001) and filtered tap water ad libitum, and maintained on a reverse 12-h light:dark cycle (lights on at 1200). Females were housed in pairs until mating. Two weeks prior to DMT exposure, estrus cycles were monitored through daily vaginal smears. Females (30/dose) were then rearranged and paired according to synchronized estrus cycles (over 4 days).

EXPERIMENT 2
Eighty-seven (n=21 control, n=22 per DMT dose groups (3, 15, and 74 ppm) timed-pregnant Sprague–Dawley (CD) female rats (Charles River, Raleigh, NC, USA), were received at gestational day (GD) 2 (sperm-positive considered gestational day 0). Upon arrival, all females were housed individually as in Experiment 1, except for the light cycle (lights on at 0600) and the temperature (19–21 °C). The total experiment was replicated over ten cohorts of dams, with treatment counterbalanced across groups, such that births occurred two to four consecutive days each week.

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dimethyl tin dichloride was dissolved in distilled/deionized water at concentrations of 0, 3, 15 or 74 mg/l (0, 1.6, 8.1, or 40 ppm Sn). Rats received the DMT or water in polypropylene/polyethylene water bottles containing double ballbearing sipper tubes. A concentrated stock solution (740 mg/l) was prepared every two weeks and stored at −20 °C; dilutions were made from this stock. Water bottles were changed and weighed twice weekly, and rats were always weighed at the same time.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted to verify the concentration and speciation of the methyl tins in the DMT solutions. The concentrated stock solution and solutions of the low and high test concentrations were sampled daily for 5 days from water bottles maintained under conditions of the animal exposure. Ion chromatography with inductively coupled plasma mass spectrometry was used to determine levels of MMT, DMT and TMT. Total tin levels were measured using inductively coupled plasma optical emission spectrometry. The limit of detection was 1 ng Sn/ml for DMT and TMT and 10 ng Sn/ml for MMT.

Total tin analysis revealed that the low concentration, as prepared, was about 10% higher than the nominal value, whereas the high concentration was only about 2% higher. The concentration of DMT did not decrease over days and neither MMT nor TMT were detected in the samples.

Details on mating procedure:

EXPERIMENT 1
After two weeks of DMT exposure, the females were bred by placing two receptive females (i.e. late-stage proestrus) with a breeder male near the end of the light cycle, and removed the next day at lights-on. Following this cohabitation period, females were individually housed, and maintained on the DMT solutions with food (Purina Formulab Chow 5008) throughout gestation and lactation. Assignment of treatment was counterbalanced across cohorts.

EXPERIMENT 2
Timed-pregnant animals were used.

Duration of treatment / exposure:

Experiment 1:
Dosing was performed before mating (2 weeks), throughout gestation and lactation (PND21).

Experiment 2
DMTC exposure occurred from gestational day 6 to day 21 of lactation.

Frequency of treatment:

daily

Dose / conc.:

0 ppm

Dose / conc.:

3 ppm

Dose / conc.:

15 ppm

Dose / conc.:

74 ppm

No. of animals per sex per dose:

Experiment 1: 30 females per dose.
Experiment 2: 22 females per dose group, 21 for control group.

Control animals:

yes

Details on study design:

EXPERIMENT 1
After two weeks of DMT exposure, the females were bred by placing two receptive females (i.e. late-stage proestrus) with a breeder male near the end of the light cycle, and removed the next day at lights-on. Following this cohabitation period, females were individually housed, and maintained on the DMT solutions with food (Purina Formulab Chow 5008) throughout gestation and lactation. Assignment of treatment was counterbalanced across cohorts.

The day of birth was designated postnatal day (PND) 0, and litters (n=13 control, 9 at 3, 74 ppm, and 10 at 15 ppm) were culled to 8 males on PND 1. In a few cases, female pups were kept to maintain equal litter sizes. On PND 21, remaining offspring were weaned and the littermates separated and housed individually on Beta-Chip bedding and provided food (Purina Rodent Chow 5001) and filtered tap water. Only male offspring, one from each litter, were tested in the different neurobehavioral tasks; each pup was evaluated in only one test. Offspring were weighed at least weekly throughout.

EXPERIMENT 2
Using the same concentrations as in Experiment 1, DMT exposure began at GD 6 and continued through gestation and lactation. Litters (n=21 control, n=22 at 3, 15 ppm, and n=20 at 74 ppm), were culled to 4 males and 4 females on PND 4. On PND 21, the offspring were weaned and the littermates separated and housed individually on Beta-Chip bedding and provided food (Purina Rodent Chow 5001) and filtered tap water ad libitum. Both male and female offspring (one from each litter) were tested in the different neurobehavioral tasks with the exception of the runway task, in which only males were tested.

Dams were weighed on the days that bottles were changed, and also on specific gestational and lactational days. All pups were weighed on specific postnatal days, and in addition, pups used for each of the neurobehavioral tests were weighed on the day of testing.

Maternal examinations:

Clinical signs, food consumption and body weight were recorded.

Fetal examinations:

RUNWAY LEARNING TEST
The runway learning test is an appetitive learning paradigm in which a food-deprived PND 11 rat pup was trained to negotiate a runway for a dry suckling reward from its anesthetized mother in the goal box. Briefly, the apparatus was a Plexiglas runway with a goal box at the end, which was maintained at 37 °C through the use of water-circulating heating pads. The pups learned to traverse the runway to reach the anesthetized dam and latency was recorded. Acquisition consisted of reinforced (R; 15 s of dry suckling) and non-reinforced (N; placement in a holding cage for 15 s) trials. If the pup failed to find the dam within the allotted time, the experimenter guided it down the runway for either (R) or to be immediately placed in the holding cage. Extinction (blocked access to the dam) immediately followed acquisition. The specific parameters for each experiment are outlined below.

Experiment 1:
Dams were anesthetized using 2 ml/kg i.p. Chloropent® equivalent. The dams were dosed approximately 15 min before testing began. Pups (n=11 control, 9 at 3 and 74 ppm, and 10 at 15 ppm) were food-deprived for 10 h prior to testing, and tested during their dark cycle. Acquisition consisted of 25 alternating (R) and (N) trials, with a maximum of 120 s for each trial and an inter-trial interval of 15 s in a holding cage. Extinction trials began on the 26th trial, and the maximum time was set at 100 s. When pups reached the criterion of two consecutive 100-second trials, they were no longer tested. All pups were tested until they met criterion, regardless of the number of trials required.

Experiment 2:
Dams were anesthetized using Nembutal sodium solution (0.65 ml i.p.; 50 mg/ml). A different training schedule was used in Experiment 2. In this paradigm, pups (n=20/dose except n=19 at 74 ppm) were food-deprived for 8 h and then tested during their light cycle. Testing began with a preliminary training session of 5 massed (R) trials, followed by a 2 min retention interval in the holding cage. There were then 25 acquisition trials in which (R) and (N) trials alternated in blocks of 5 trials, beginning and ending with 5(R) trials, with an 8-second inter-trial interval. The maximum time allowed for each trial was 100 s. Extinction trials began on the 26th trial, and the criterion to extinction was one trial with a 100-second latency. A maximum of 10 extinction trials were run.

MOTOR ACTIVITY
Motor activity data were collected using automated figure-eight chambers. Photocell interruptions (counts) were recorded over 5-min intervals of the 30-minute test session. In Experiment 1, motor activity was assessed in males at PNDs 13, 17 and 21 (n=14 control, n=9 at ppm, n=10 at 15 ppm, and n=9 at 74 ppm). In Experiment 2, only PND 17 male and female offspring (one male and one female from each litter; n=21 control and 15 ppm, n=20 at 3 ppm, and n=17, 74 ppm) were tested.

SPONTANEOUS ALTERATION (Experiment 2 only)
Spontaneous alternation was measured on PND 25 using a Plexiglass T-shaped apparatus. Although others have not reported adult levels of alternation (85–90%) until one month of age or older, pilot studies in our laboratory indicated that in our two-choice alternation task, using a freechoice, continuous exploration protocol, pups achieved 80% alternation by PND 25. Thus, in the present study, PND 25 pups were placed in the stem for a 30 s acclimation, after which time the gate was raised allowing the rat to enter either arm. The rat was then allowed to explore freely between only the two
opposing arms for 5 min. All arm entries were counted as the measure of motor activity, whereas alternation was considered when the rat left one arm and entered the other. A minimum of six arm entries was required to calculate alternations to avoid erroneous data due to low activity. Percent alternation was calculated as the number of opposite arm entries divided by the total arm entries less one (to subtract the first entry). Both males and females were tested at 10/dose.

MORRIS WATER MAZE (Experiments 1 and 2)
Spatial memory was evaluated using a Morris water maze. Rats were tested as adolescents/young adults (beginning about 7 weeks old in Experiment 1, 12 weeks old in Experiment 2). Each test trial was videotaped and the image digitized for computer analysis using maze-tracking software. Dependent variables included swim speed, latency and path length to find the platform, and time spent in the outer edge of the tank or one of the three concentric zones.

For spatial training, rats learned the fixed position of the platform during 2 trials a day with an inter-trial interval of 5 min, for 9 days. The starting position was semi-randomly varied (all four starting positions were used before one was repeated, but the order itself never repeated) every 2 days. The maximum trial time was 60 s, after which time the observer guided the rat to the platform. On the 10th day, a probe trial was conducted in which the platform was removed and the subject's tendency to search in the correct quadrant was measured over 60 s. Dependent variables were the Gallagher proximity score, and percent total time within each quadrant. A visible probe trial was also conducted using a raised platform of a contrasting colour to confirm that the tested animals were not visually impaired.

In Experiment 1, only males were tested in the water maze (n=11 control, 7 at 3 ppm, 9 at 15 ppm, and 11 at 74 ppm) whereas both males and females (one from each litter) were tested in Experiment 2 (n=10/sex/dose).

NEUROPATHOLOGY (Experiments 1 and 2)
Experiment 1:
For neuropathological evaluations, male rats (n=6–8/dose at PND1, n=5–8/dose at PND 12, n=5–9/dose at PND22, and n=5/dose at adult age, 80–90 days old) were deeply anesthetized with pentobarbital and perfused via the left ventricle with buffered 4% formaldehyde:0.1% gluteraldehyde. Sagittal blocks of tissue were embedded in paraffin and sectioned to include all major structural landmarks of the brain in each section (e.g., olfactory bulb, striatum, cerebral cortex, hippocampus, thalamus, hypothalamus, brainstem, cerebellum). Twenty-four sections of brain from each rat, from each age, at each dose, were stained with hematoxylin and eosin.

Brains from all control and high-dose rats at all ages were evaluated by a certified pathologist. Step-down assessments, i.e., evaluation of the lower dose groups, were only conducted in the adult rats due to the remarkable findings in the high-dose group. Scoring of the severity of observed changes was conducted with the pathologist blind to the dosage group.

Experiment 2:
Brains were prepared and examined as described for Experiment, but only adult rats (both males and females) were used (n=10/dose/sex, except n=9 15 ppm males).

BRAIN WEIGHTS (Experiments 1 and 2)
Male rats were decapitated under CO2-induced anesthesia at PND1, 12, 22, and as adults (Experiment 1; n=4–11/dose/age) or PND12, 22, and as adults (Experiment 2; n=7–9/dose/age). From PND12 on, all subjects came from different litters. Brains were quickly removed and weighed.

APOPTOSIS ASSESSMENT
Apoptosis was quantified using a Cell Death ELISA procedure. Modifications to the procedure in the kit have been described previously; the kit has been adapted for use with intact tissue and validated with both fresh and frozen brain tissue. Moreover, the results have been corroborated qualitatively by agarose gel and TUNEL data. In short, the enzyme-linked immunosorbent assay (ELISA) uses antibodies to bind fragmented DNA characteristic of apoptotic cell death. The bound fragments (i.e. nuclesomes) are then quantified photometrically.

Experiment 1:
After weighing, brains were dissected free-hand into the following regions: brainstem, neocortex, hippocampus and cerebellum. Each region was immediately frozen in 2-methyl butane on dry ice for 30 s and stored at −70 °C [30]. ELISA assays were conducted only on tissues collected at PND22 (n=3–7/dose/region) and as adults (n=2–4/dose/region).

Experiment 2:
Tissues were collected from male rats only as described for Experiment 1 on PND12, 22, and as adults, with n=6–8/dose/region.

Statistics:

Where data were collected from littermates, they were analysed using litter as the unit (e.g., sex or multiple observations nested within litter). Continuous data (e.g., body weight, water intake, activity counts, etc.) were analysed using a general linear model ANOVA. Extinction in the runway test was analysed with a survival model for censored data and count data (e.g., number of pups learning, pregnancy rate) were compared using Fisher's exact test. In the second runway study, within-subject trend analyses (SAS) of the slopes of each 5-trial block were used to evaluate whether the latencies were increasing or decreasing (significantly different from a slope of zero) over the 5 trials, or whether there was no change (slope of zero). Whenever the same rat was used in repeated tests (e.g., repeated motor activity testing, body weights over time, within-session activity), the analyses included time (or interval) as a repeated factor; however, if no interaction between time/interval and treatment was revealed, data were collapsed across time. Likewise, if there was a significant interaction between sex and dose (or sex, dose, and age/time), only then were males and females analysed separately to evaluate dose (and/or age/time) effects. Where sex interactions were not significant, the data were combined. Dunnett's t-test was used to compare dose groups with the control. In all cases, resulting twotailed probability values ≤0.05 were considered significant.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Experiment 1: Only the high concentration altered maternal weight gain, which was significantly lower than control throughout exposure.

Experiment 2: Reductions in body weight were not evident until lactation, at which time the high-concentration body weight was significantly lower than controls.

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Experiment 1: Overall, water consumption increased among all dose groups throughout gestation and lactation. In the first two weeks of exposure (premating), consumption was significantly decreased in all DMT-exposed groups. During the first half of gestation, only the high concentration decreased consumption. Although the high-concentration consumption appeared lower throughout the rest of exposure, these differences were not significantly different from control.

Experiment 2: From the beginning of exposure to the end of gestation, fluid intake was significantly lower in the 15 and 74 ppm dose groups. Additionally, the 3 ppm dose group showed decreased consumption during the second week of exposure. Intake returned to control levels in all but the high-concentration group during lactation.

Details on maternal toxic effects:

Reproductive performance:
Comment: The two experiments cited below utilized the same doses [0, 3, 15, 74 ppm] and the same route of exposure and vehicle [oral via drinking water], but they utilized clearly different dosing regimens. This discrepancy in the experimental designs introduces a degree of uncertainty when attempting to compare the results from the two studies. Experiments 1 and 2 are not exact replicates.

Experiment 1 incorporated a 2 week pre-mating dose period, a gestational exposure from gestational day [GD] 0 through birth at GD21, exposure during lactation from post-natal day [PND] 0 through weaning at PND21. Total maternal exposure was 56 days and total offspring exposure was 42 days.
Experiment 2 did not have a 2 week pre-mating dose period, gestational exposure did not begin until GD6 and continued through birth at GD21, exposure during lactation from post-natal day [PND] 0 through weaning at PND21 was the same as in Experiment 1. Total maternal exposure was 36 days and total offspring exposure was 36 days.

Data:
Experiment 1: The overall pregnancy success rate was low (47 of 120 rats, 39%). There was no treatment effect on the number of pregnancies: n=10 control, 14 at 3 and 15 ppm, and 9 at 74 ppm. All births occurred within a 24-hour time frame and there was no effect of treatment on the number of pups per litter which supports the conclusion that maternal gestational integrity was unaffected by DMTC.

Experiment 2: All of the timed-pregnant females delivered with the exception of one 74 ppm DMT female. Additionally, one 74 ppm female delivered only six pups and was not used. All of the deliveries occurred when expected and there was no effect of treatment on the number of pups per litter which further supports the conclusion that maternal gestational integrity was unaffected by DMTC.

CONCLUSION ON MATERNAL TOXIC EFFECTS
Despite the differences in design between Experiments 1 and 2, DMTC produced maternal toxicity at the high dose [74 ppm] in both segments.

Dose descriptor:

NOAEL

Effect level:

15 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

water consumption and compound intake

Remarks on result:

other: (average intake 1.75 mg/kg bw/day study 1 or 2.1 mg/L study 2)

Abnormalities:

effects observed, treatment-related

Localisation:

other: general maternal toxicity

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects
> Offspring number
Experiment 1: The total numbers of live pups per litter were (mean ±SEM): control, 14.4±1.0; 3 ppm, 13.3±0.7; 15 ppm, 15.0±0.9; and 74 ppm, 12.8±0.8. Statistical analysis suggested a trend toward fewer males in the treated groups (dose F(3, 43)= 2.35), however the p value [p=0.085] was not less than 0.05 , and there there was no significant treatment-by-sex interaction. The suggested trend may have been due to the three litters in the high-concentration group which had one to two dead pups, whereas control litters had none; however, there were no statistically significant differences in litter size across groups. There were no external anomalies reported in controls or in any treatment group. After culling at PND1, three litters (one control, two at 74 ppm) lost three to four more pups each, again not a difference of biological significance.

Experiment 2: The total number of live pups per litter was not significantly different across groups, thus diminishing the relevance of the suggested trend observed in Experiment 1. There were no external anomalies reported in controls or in any treatment group.

Conclusion: These data support the conclusion that DMTC was not embryotoxic in either experiment and that the gestational integrity of the conceptus was unaffected by DMTC at any dose.


> Offspring postnatal growth
Experiment 1: There were no statistically significant differences in (male) pup weights across groups at any postnatal period or when pups were weighed prior to each of the neurobehavioral tests. Female pups were maintained to preserve equal litter sizes across groups, but female pup weights were not reported. It is inferred that differences would have been reported had they occurred.

Experiment 2: Body weight changes among the pups during the lactation period showed a significant dose-by-sex interaction (F(3, 69)=3.0, p=0.037). Step-down analyses of males and females revealed that males in the high-concentration group weighed significantly less than controls throughout lactation, and the decrease in females in the same group reached significance only at PND 17 and 21. This effect was clearly transient. There were no treatment-related effects on body weight which was measured weekly after weaning, nor was there an effect at any time when offspring were weighed prior to neurobehavioral testing.
These data are equivocal and confounded by the experimental design. A statistical difference in body weights of the high-dose offspring was observed only after birth and only in Experiment 2, the segment with the shorter exposure period. This was not an in utero effect. The observed differences were transient and it cannot be determined with certainty whether this observation was a direct effect of postnatal exposure of the neonates in Experiment 2 or a result of poor maternal-neonatal interaction in the second experiment.

Conclusion: These data support the conclusion that DMTC was not fetotoxic in either experiment at any dose. In addition, an observed decrease in neonatal body weight of the high dose pups during lactation was equivocal and transient. Body weights for all treated groups of offspring recovered to normal after weaning.

> Runway testing
Experiment 1: Using a criterion of having at least one latency less than the maximum time of 120 seconds, several pups in each treatment group failed to learn the task; however, there was no treatment-related difference in the incidence of non-learners (control, 3 of 12; 3 ppm, 1 of 9; 15 ppm, 0 of 10; and 74 ppm, 1 of 9). In the extinction phase, the median number of trials to reach the criterion (two consecutive trials of 100 seconds) for each dose group was: control, 18; 3 ppm, 13; 15 ppm, 15.5; and 74 ppm, 24. Again, there was no reported treatment-related difference in the outcomes.

> Experiment 2: This segment used the massed-alternation paradigm and the same criterion as above for learning success of the runway task. The proportion of pups that failed to learn were control, 3 of 20; 3 ppm, 5 of 20; 15 ppm, 6 of 20; and 74 ppm, 6 of 19. There were no statistically significant differences in these outcomes. Analysis of the within-subject slopes (linear trend analysis) for each 5-trial block was much less variable. Learning, as defined by decreasing latencies during the reinforced blocks, gives a negative slope, whereas extinction, or increasing latencies (or no change in latency) during non-reinforced blocks, gives either a positive or zero slope. Such a pattern shows the contingency control over the behavior. Both the control and low-dose groups showed significant decreasing slopes (p'sb0.04 for all) for each of the set of R-trials, and the slopes during N-trials were not different from zero. In contrast, the 15-ppm group did not show decreased latencies on any of the R-trials, and the only significant slope change (p=0.006) was in the first N-trial (increasing latencies). The high-dose group showed a decreasing slope (p=0.008) only during the last block of R-trials. Thus, learning was not apparent during any R-trial blocks in the 15-ppm group, and was only achieved at the last set of trials in the 74-ppm group.
A number of pups did not extinguish the behavior in 10 trials, and therefore did not meet the criterion for extinction: control, 4 of 17; 3 ppm, 2 of 15; 15 ppm, 3 of 14; and 74 ppm, 1 of 13. Thus the expected outcome, extinction of the behavior, did not occur in 23.5%, 13.3%, 21.4%, and 7.7% of the pups for the control, low, mid and high dose groups, respectively. While this pattern of unlearning the task appears to suggest the inverse of the learning pattern, there were no statistically significant differences in these outcomes.

Conclusion: DMTC had no consistent statistically significant effect on the development of runway learning as a behavioral maturation parameter.

> Motor activity
Experiment 1: Total motor activity counts during 30 minute sessions increased with the age of the animals when tested on PND 13, 17 and 21, however there were no treatment-related differences in the activity levels. Analysis of the within-session activity (in 5-min intervals) showed that habituation (significant change in activity across intervals) was not evident until PND 21 in all treatment groups. This is not a treatment-related adverse outcome.

Experiment 2: Total counts for each group (sexes combined, mean ±SEM) were: control, 115.4±7.8; 3 ppm, 114.2±5.6; 15 ppm, 121.5±9.2; and 74 ppm, 119.0±8.8). Unlike in Experiment 1, habituation was evident in all treatment groups at PND 17, but this is not a treatment-related adverse outcome.

Conclusion: These data support the conclusion that DMTC had no effect on the development of motor activity as a behavioral maturation parameter.

Spontaneous alternation (Experiment 2 only)
The percent of alternations showed no significant difference across groups or gender. It can be concluded that DMTC had no effect on the development of spontaneous alternation as a behavioral maturation parameter.

> Morris water maze
[This is an integrated behavioral assessment which includes assessment of maze learning and swim speed, and an evaluation of test animals’ search parameters]
Experiment 1: All groups eventually learned; however, the middle dose group showed significantly longer latencies during the first week of training (dose F(3, 31)=3.57, p=0.025). Analyses of the spatial distribution of swimming indicated that, in the second week, the low and middle dose groups spent significantly less time in the middle zone (dose F(3, 31)=5.79, p=0.003), and in addition, the 15 ppm group spent more time in the outer zone (dose F(3, 31)=5.52, p=0.004). This represented a less efficient search strategy, since the platform was located in the middle zone, and may have influenced the longer latencies observed in this treatment group. The high dose group showed no differences on any of these parameters.
In this segment swim speed was not affected either positively or negatively by treatment with DMTC at any dose. In this segment neither the memory probe search parameter [platform removed after its location is learned] nor the visual probe search parameter [platform raised above water surface to facilitate its acquisition as a target] were affected by treatment with DMTC at any dose.

Experiment 2: As in Experiment 1, the middle dose group had significantly higher latencies to learn the platform position (dose F(3, 71)=3.1, p=0.032). This was seen in both sexes in the second week, as evidenced by the lack of a treatment-by-sex interaction. There were, however, differences between the sexes in terms of the spatial pattern of swimming (first week, middle zone block-by-trial-by-dose by-sex F(12, 284)=2.03, p=0.022; outer zone block-by-trial by-dose-by-sex F(12, 284)=1.87, p=0.038). In the first week of training, females spent less time in the middle zone, and more time in the outer zone. On days 2 and 3, this pattern was significant in all dose groups. This propensity for the outer zone persisted in the middle dose group into the second week of training (dose F(3, 71)=7.44, p=0.0002), and was significant for both males and females. In both cases, the 15 ppm rats spent more time searching in the outer zone, and less time in the middle zone of the tank.
Analysis of the memory probe again revealed less time in the middle zone (dose F(3, 71)=3.21, p=0.028), in both males and females of the middle dose group. The time in the quadrants differed (dose-by-quadrant F(3, 71)=4.22, p=0.008), with the low dose group showing significantly less time in the correct quadrant, and the middle dose group showing a similar trend (mean % time in correct quadrant ± SE: control, 48.3±2.2%; 3 ppm, 39.7±3.0; 15 ppm, 40.6±2.2%; 74 ppm, 48.6±2.2%).
In this segment, as before, swim speed was not affected either positively or negatively by treatment with DMTC at any dose. The visual probe search parameter was not affected by treatment with DMTC at any dose. Outcomes for the memory probe search parameter were not reported.

Conclusion: These data support the conclusion that in utero and postnatal exposure to DMTC did not affect the development of integrated behavioral assessments which included assessment of maze learning and swim speed. The evaluation of test animals’ search parameters caused significantly higher latencies to learn the platform position in both sexes at the mid dose only. The lack of any significant differences at either a five-fold higher dose or at a dose one-fifth of that producing the outcome suggest the result may be anomalous and should be classified as equivocal. The lack of a dose-response in two separate and similar experiments supports the conclusion that in utero and postnatal exposure to DMTC did not consistently effect the development or maturation of the integrated neurobehavioral assessments or the test animals’ search capabilities.

> Neuropathology
Experiment 1: Histopathological alterations in the brain of offspring of dams exposed to DMT were noted in the cerebral cortex of rats sacrificed at PND22 and as adults. Three of five (60%) adult offspring at 74 ppm and one of five (20%) PND22 rats at 74 ppm had minimal/slight vacuolation of the neuropil of the gray matter of the cerebral cortex. Step-down evaluations of the lower dose groups showed similar vacuolation at 15 ppm (1 of 5 adults) and 3 ppm (1 of 5 adults). There were no lesions in the offspring at PND1 or 12, or in the offspring at the lower doses at PND22. The cerebral cortical lesion was characterized by 2–4 micron diameter, round vacuoles in the gray matter neuropil in the region of the orbital cortex. On a score of 1 (minimal) to 5 (severe), the rats in the lower dose groups received scores of 1, whereas the high-dose rats received scores of 2 (slight/mild).

Experiment 2: One male offspring at 74 ppm had a single neuron in the midbrain with central chromatolysis. The significance of this finding in a single neuron in a single treated rat remains undetermined.

Conclusion: The lack of neural histopathological anomalies in Experiment 2 suggest the results from Experiment 1 should be classified as equivocal.

> Brain weight
Experiment 1: Analysis of brain weights revealed an overall effect of dose (F(3, 88)=3.61, p=0.016) but no interaction with age.
The data showed significant decreases in the low- and high dose groups when collapsed across the ages; overall, the low dose was 4% lower, and the high dose 8% lower, than controls. The mid dose group average was equal to the control mean.

Experiment 2: As in the first experiment, there was an overall effect of dose (F(3, 67)=4.05, p=0.01) in male rats but no interaction with age. Collapsed across age, only the high dose group showed a significant decrease of 4%.

Conclusion: Brain weights were recorded from animals at four different ages [PND1, 12, 22, and as adults] in Experiment 1. In Experiment 2 brain weights were recorded at three different ages [PND12, 22, and as adults]. The analysis collapses these into a single assessment of brain weight. These data support the conclusion that exposure to DMTC may adversely affect brain weight, however the effect cannot be clearly ascribed to a developmental effect because DMTC is known to be neurotoxic in adult animals and the observations in these experiments could be the result of postnatal exposure alone.

> Apoptosis
Experiment 1: Significant dose-by-age interactions were observed for the cerebellar (dose-by-age F(3, 27)=2.93, p=0.05) and cortical (dose-by-age F(3, 27)=5.79, p=0.003) DNA fragmentation data, which were then determined to be significant decreases only at PND22 in the cerebellum (15 and 74 ppm) and cortex (all doses). These changes, however, did not show a clear dose–response. No differences in DNA fragmentation were observed in any of the brain regions for adult animals.

Experiment 2: A dose-by-age interaction (F(6, 60)=11.90, pb0.0001) for the brainstem data presented no treatment effect at PND12, with significant increases relative to control at PND22, and decreases in adults. These significant effects were seen in the mid and high dose groups. These changes may represent a shift in the normal decrease observed in control rats. In addition, cerebellar data revealed a small but significant increase (dose-by-age F(6, 60)=3.36, p=0.006) in DNA fragmentation at PND12, but only at the high dose (means ±SE: control, 1.0±0.07; 3 ppm, 1.30±0.07; 15 ppm, 1.06±0.04; 74 ppm, 1.07±0.08).

Conclusion: These data are inconsistent on three levels: location, direction and age. Brain apoptosis was recorded for four different brain regions [brainstem, neocortex, hippocampus and cerebellum] and from animals at two different ages [PND 22, and adult] in Experiment 1. In Experiment 2 brain apoptosis was recorded for the same four brain regions and from animals at three different ages [PND 12, 22, and adult]. The significant differences observed in Experiment 1 were decreases in the cerebellum and cortex, and only at PND22, not in adults. Whereas the significant differences observed in Experiment 2 were: in brainstem [PND22] where the difference was an increase; in cerebellum [PND12] where the difference was an increase; and in brainstem [adults] where the difference was a decrease. NO differences were observed in cortex.

The interpretation of these results is confounded by the normal decrease in apoptosis known to occur in control rats, a progression which was confirmed in this study. Overall, these data support the conclusion that exposure to DMTC may affect brain apoptosis, however the effect is inconsistent and cannot be clearly ascribed to a developmental effect because DMTC is known to be neurotoxic in adult animals and the observations in these experiments could be the result of postnatal exposure alone.

Dose descriptor:

NOAEL

Effect level:

15 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Brain lesions

Abnormalities:

effects observed, treatment-related

Localisation:

other: Brain lesions

Developmental effects observed:

yes

Lowest effective dose / conc.:

15 ppm

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: DMTC intake (mg/kg/day) calculated from average fluid consumption and body weights for each phase of Experiments 1 and 2

	Concentration (ppm)	Intake (ml/d)	Weight (g)	DMTC (mg/kg/d)
Experiment 1
Pre-mating	0	27.6	266.1	-
	3	24.1^a	255.0	0.28
	15	20.4^a	263.3	1.16
	74	14.7^a	248.6^a	4.38
Gestation	0	37.5	332.4	-
	3	34.7	321.0	0.32
	15	34.1	331.4	1.54
	74	29.6^b	307.2^a	7.13
Lactation	0	61.9	339.5^a	-
	3	58.0	336.2	0.52
	15	58.3	342.7	2.55
	74	50.3	304.2^a	12.2
Experiment 2
Gestation	0	36.8	301.0	-
	3	32.1^b	296.3	0.33
	15	30.1^a	295.8	1.53
	74	28.2^a	287.6	7.26
Lactation	0	62.8	324.3	-
	3	60.9	318.3	0.57
	15	56.7	318.3	2.67
	74	47.6^a	297.1^a	11.9

 

^aIndicates data significantly different from control throughout the period measured.

^bIndicates data significantly different from control during part of the period measured.

Conclusions:

The results support the following conclusions with respect to the effects of perinatal exposure to DMTC:
- maternal toxicity expressed as decreased maternal fluid consumption and depressed maternal weight gain at the high concentration (74 ppm) [in both DNT studies];
- no treatment-related embryotoxicity measured as gestational integrity [in both studies];
- decreased postnatal growth only during lactation with evidence of F1 recovery thereafter [Study 2 only];
- no significant effect on either of two memory probes [in both studies];
- altered the spatial learning in the Morris water maze, but only at the intermediate concentration (15 ppm) [no dose-response in either study];
- decreased F1 brain weight [consistent only at a maternally toxic dose (74 ppm) in both studies];
- inconsistent and minimal to slight histopathological alterations in F1 brain, characterized as vacuolation of the cortex [in Study 1 only at high dose on PND22 and all doses as adults, no findings in Study 2 at any dose];
- inconsistent altered levels of an apoptotic marker (DNA fragmentation) [in Study 1 decreased at PND22 at all doses but not in adults at any dose; in Study 2 no differences at PND12 at any dose, increased at PND22 at mid and high doses but decreased in adults at the same dose levels].
Comment: The age and brain area at which this was seen was not consistent and no effect was consistent across the two studies or at any dose.

Executive summary:

A limited number of in vitro and in vivo studies suggest that Dimethyltin Dichloride (DMT) produces neurotoxicity. The DNT studies were initiated to evaluate long-term neurobehavioral changes in F1 animals following perinatal exposure. In the first study, female Sprague–Dawley rats were exposed 2 weeks prior to mating and throughout mating, gestation and lactation. Male offspring were compared to controls for differences in: 1) pre-weaning learning in an associative runway task, 2) motor activity ontogeny, 3) spatial learning and retention in the Morris water maze as adults, 4) brain weight, 5) biochemical evidence of apoptosis, and 6) neuropathology. Maternal toxicity was expressed as decreased maternal weight gain. Only In the first of the two studies was developmental toxicity expressed as minimal to slight brain histopathology vacuolization of F1 adults at all doses, decreased brain weights, and one of several neurobehavioral end points was altered.

In the second study, exposure to timed pregnant Sprague–Dawley rats occurred via drinking water from gestational day 6 to weaning and a similar battery of behavioral tests was administered to the F1 animals. The high concentration again depressed maternal weight gain. The F1 animals at the high dose had lower weight only during lactation, not thereafter. There was decreased brain weight but no correlating adverse histopathological findings in high-dose offspring. No significant differences in motor activity or in the spontaneous alteration activity test occurred as the result of treatment. Statistically significant learning deficits were not observed in the runway test, but a difference from controls was observed in mid-dose animals only in the water maze test.

The results of both studies demonstrate a lack of dose-response in spatial learning after perinatal DMT exposure. Changes in brain weight, and the occurrence of neuropathological lesions in offspring at the high dose indicate a potential for neurotoxicity of DMT at a maternally toxic dose. Histopathological alterations of minimal to slight severity in the brain of offspring of dams exposed to DMT in Experiment 1 were noted in the cerebral cortex of rats sacrificed at PND22 and as adults. These lesions were not observed in Experiment 2 in any brain region.

The NOAEL was determined to be 15 ppm (equivalent to 1.16-2.67 mg/kg/day).

These results provide some evidence that postnatal developmental toxicity can be produced by perinatal exposure to DMT, but only at maternally toxic doses of DMT. These results can be compared to earlier findings in two additional developmental neurotoxicity studies from the same laboratory with monomethyl tin [MMT] in which the results indicated that perinatal exposure to MMT, even at concentrations which decreased fluid intake, did not result in significant neurobehavioral or cognitive deficits in the F1 animals and no behavioral manifestations of developmental neurotoxicity. Thus, the evidence that methyltin compounds produce developmental neurotoxicity following perinatal exposure via maternal drinking water is equivocal and is marginally sufficient for classification.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across to structurally similar substance dimethyltin dichloride (DMTC) (EC Number 212-039-2, CAS Number 753-73-1), see attached justification.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

dose level:

Effect level:

74 mg/L drinking water

Based on:

test mat.

Basis for effect level:

body weight and weight gain

water consumption and compound intake

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/L drinking water

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

3 mg/L drinking water

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

dose level:

Effect level:

74 mg/L drinking water

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: neuropathology; brain weight; apoptosis

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/L drinking water

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: neurobehavioral, apoptosis

Key result

Dose descriptor:

NOAEL

Effect level:

3 mg/L drinking water

Based on:

test mat.

Sex:

male/female

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Reason / purpose for cross-reference:

other: read across: target

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

refer to "Principles of method if other than guideline" for further information

Principles of method if other than guideline:

The method followed the basic general outline of OECD Guideline 414, however there were several deviations.
The most notable of these were:
- Dosing occurred in the period of organogenesis (days 7 – 17 of gestation)
- Group size was below that recommended in the guideline (Study I, 5 groups of 10; Study II, 8 groups of 8 – 11)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan Inc. (Tokyo, Japan)
- Age at study initiation: 3 months
- Housing: Animals were individually housed.
- Diet (e.g. ad libitum): ad libitum. Fed commercial laboratory chow (NMF; Oriental Yeast Co., Ltd. Tokyo, Japan)
- Water (e.g. ad libitum): ad libitum tap water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%):60 ± 20% relative
- Photoperiod (hrs dark / hrs light): Constant day/night cycle (lights on from 7:00 to 19:00 hr)

Route of administration:

oral: gavage

Vehicle:

other: saline

Analytical verification of doses or concentrations:

no

Details on mating procedure:

A female rat was paired overnight with a male of the same age and the day on which sperm was observed in the vaginal smears was designated as day 0 of gestation.

Duration of treatment / exposure:

Two teratological studies were performed. In the first (Study I), DMTC was administered to pregnant animals during the organogenetic phase of gestation (days 7-17 of gestation). DMTC was administered to pregnant females at 0, 5, 10, 15 or 20 mg/kg/day on days 7-17 of gestation. The volume of vehicle was held constant at 2 mL/kg based on maternal body weight.

In the second teratological study (Study II), DMTC was administered to pregnant animals at 0, 20 or 40 mg/ kg/day for two to three consecutive days at one of four different periods of gestation (gestational days 7-9, 10-12, 13-15 and 16-17). The group treated with DMTC at 20 mg/kg/day on days 16-17 was not tested. Control animals were administered an appropriate volume of vehicle without DMTC on days 10-12 of gestation.

Duration of test:

20 days

No. of animals per sex per dose:

Study I: Mated females were randomly assigned to five groups of ten rats each.
Study II: Mated females were randomly assigned to eight groups of 8-11 rats each.

Control animals:

yes, concurrent vehicle

Details on study design:

All procedures were substantially the same in the two teratological studies. Animals were randomly assigned to test groups.

Maternal examinations:

Maternal body weight and food intake were measured daily. Pregnant females were observed daily for clinical signs of toxicity and were sacrificed by overdose of ether anesthesia on day 20 of gestation. Maternal thymus and brain weights, and the gravid uteri weights (only in study II) were also recorded.

Ovaries and uterine content:

The position and the number of living fetuses and implantation loss in the uterus, and the number of corpora lutea, were recorded. Uteri with total resorption were isolated and stained with 10% ammonium sulfide to determine the total number of implantations.

Fetal examinations:

The living fetuses were examined for their sex and external malformations, and were then weighed. Half of the living fetuses in each litter were fixed with 95% ethanol and processed for staining of the skeleton by the alizarin red S dye method. These preparations were examined for skeletal abnormalities. The other half of each litter was fixed in Bouin's solution and examined for visceral abnormalities according to the method of Wilson.

Statistics:

Data on the number of dams with living fetuses and with total resorption and the number of malformed fetuses were analysed by Fisher's exact test. Other data were analysed by Dunnett's multiple comparison method) in a parametric or non-parametric manner. The litter was used as the statistical unit for calculation of fetal values.

Food efficiency:

not examined

Details on maternal toxic effects:

Details on maternal toxic effects:
STUDY I
Oral treatment of pregnant rats with DMTC during the organogenetic phase of gestation (days 7-17 of gestation) caused reduction in the maternal body weight gain in a dose dependent manner. Significantly reduced body weight gain was observed in pregnant rats treated with DMTC at 15 or 20 mg/kg/day in late gestation. Maternal food in-take was significantly reduced by DMTC-treatment at 20 mg/kg/day, but no other statistically significant differences in maternal food intake were present.

Oral administration of DMTC at 20 mg/kg/day resulted in the death of two pregnant rats, one died on day 18 of gestation and another died on day 19 of gestation. These deaths were considered to be DMTC treatment related because the animals exhibited severe clinical signs of toxicity (piloerection, ataxia, perinasal and periocular staining, vaginal bleeding, tremor and convulsion) for about four days prior to their deaths. No gross pathological changes were noted upon necropsy in the organs of the dead dams. No other mortalities were observed in either the control or DMTC-treatment groups.

Perinasal and periocular staining, piloerection and ataxia were observed in all the pregnant rats administered DMTC at 20 mg/kg/day mainly after day 15 of gestation. In addition to these clinical signs of toxicity, vaginal bleeding, tremor and convulsion were observed in three pregnant animals other than two dead pregnant rats in the late stage of gestation. No clinical signs of toxicity were observed in the other groups. Maternal thymus weights were reduced in a dose-dependent manner with significance at 15 and 20 mg/kg/day on day 20 of gestation. Maternal brain weight was unaffected in any group.

Total resorption was observed in one of eight living pregnant rats at 20 mg/kg/day. This same rat also exhibited vaginal bleeding, tremor and convulsion in the late stage of gestation. Mean body weight in living fetuses of both sexes decreased in a dose-dependent manner with significance at 15 and 20 mg/ kg/day. No significant differences were noted in the number of corpora lutea, implants and living fetuses, and the incidence of post-implantation loss and sex ratio.

STUDY II
Oral gavage administration of DMTC at 40 mg/ kg/day to pregnant rats on days 10-12 of gestation caused significant reductions of maternal body weight gain on days 13, 16 and 17 of gestation and reduction of food intake on the consecutive days of gestation after day 12 of gestation. No significant reduction in maternal body weight gain was observed in other treatment period groups, in spite of the reduced food intake caused by the treatment. There were no significant differences in general behaviour among the groups including controls.

Maternal thymus weights and adjusted body weight gain were reduced significantly in the group treated with DMTC at 20 mg/kg/day on days 10-12 of gestation and in every treatment period groups at 40 mg/kg/day. Gravid uterus and maternal brain weights were unaffected at either dose level in any treatment period group. Total resorption was observed in one of 10 dams in the group treated with DMTC at 40 mg/kg/day on days 7-9 of gestation. In this group, mean fetal body weight of the females was reduced. No significant differences were noted in the number of corpora lutea, implants and living fetuses and incidence of post-implantation loss, sex ratio and male fetal body weight at either close level in any treatment period group.

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

other: thymus atrophy

Abnormalities:

effects observed, treatment-related

Localisation:

other: general maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
STUDY I
The incidence of external malformations increased in fetuses from dams exposed to DMTC at 20 mg/kg/day from days 7-17 of gestation. There were 21 fetuses with cleft palate from five of seven pregnant rats with living fetuses on day 20 of gestation. In addition to cleft palate, one fetus was associated with general edema and pes varus and one with general edema. There were two fetuses with omphalocele from one dam exposed to DMTC at 15/mg/day, but the incidence was not statistically significant. No other external malformations were observed in either the control or DMTC-treatment groups.

No significant difference was observed in the incidence of skeletal malformations and skeletal variations. No significant difference was observed in the incidence of visceral malformations, however the number of visceral variation, dilation of the renal pelvis, was significantly increased at 20 mg/kg/day.

STUDY II
The incidence of external, skeletal and visceral malformations did not significantly increase at either close in any treatment period group. No cleft palate were found in any fetus treated with DMTC at 20 or 40 mg/kg/day on two to three consecutive days at one of four different treatment periods of gestation. The numbers of fetuses with skeletal variation and visceral variation increased significantly at 40 mg/kg/day in some treatment periods. Numbers of fetuses with cervical ribs increased in the groups treated with DMTC on days 7-9 or 13-15 of gestation. Numbers of fetuses with splitting of first cervical vertebral arches and with kinked ureter increased in the group treated with DMTC on days 7-9 and 16-17 of gestation, respectively.

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: rib

skeletal: vertebra

other: external (cleft palate); visceral/ soft tissue: urinary (dilation of the renal pelvis)

Developmental effects observed:

yes

Lowest effective dose / conc.:

15 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1. Effects of DMTC on Maternal Thymus and Brain Weights on Day 20 of Gestation

	Saline 2 mL/kg	DMTC (mg/kg)
		5	10	15	20
Body wt. (g)   Thymus wt. (mg)   Brain wt. (g)	333 ± 26.7   169 ± 37.5     1.75 ± 0.02	334 ± 21.9   190 ± 32.6     1.72 ± 0.05	321 ± 21.0   168 ± 26.8     1.71 ± 0.07	315 ± 9.2   151 ± 13.6*     1.76 ± 0.04	252 ± 41.1**   45 ± 21.7**    1.68 ± 0.04

Pregnant rats were treated orally with DMTC on days 7-17 of gestation. These weights were recorded on day 20 of gestation. Values are mean ± S.D. *Significantly different from control, p < 0.05. **Significantly different from control, p < 0.01.

 

 

Table 2. Effects of DMTC Exposed to Pregnant Rats on Two or Three Consecutive Days at One of Four Different Treatment Periods of Gestation, on Maternal Brain, Thymus, Gravid Uterus Weights and Adjusted Body Weight Gain at the Day 20 of Gestation

	Saline days 10-12 2 mL/kg	DMTC (mg/kg)
		days 7-9		days 10-12		days 13-15		days 16-17
		20	40	20	40	20	40	40
Body wt. (g)   Brain wt. (g)   Thymus wt. (mg)   Body weight gain (g)   Gravid uterus wt. (g)   Adjusted body weight gain^a(g)	338 ± 19.7   1.79 ± 0.08   253 ± 45.1   112 ± 21.6     65 ± 20.0      47 ± 7.0	330 ± 23.2   1.79 ± 0.04   220 ± 22.7   110 ± 12.5     69 ± 10.2      40 ± 7.4	330 ± 47.4   1.80 ± 0.05   208 ± 37.6*   110 ± 13.1     75 ± 11.7      35 ± 6.0**	325 ± 23.1   1.79 ± 0.04   207 ± 39.9*   107 ± 18.0     72 ± 11.1      35 ± 9.6*	312 ± 22.7   1.76 ± 0.06   190 ± 36.9**   95 ± 17.8     66 ± 19.7      30 ± 9.2**	331 ± 12.7   1.75 ± 0.06   217 ± 15.3   111 ± 9.0     69 ± 4.5      42 ± 6.5	325 ± 14.8   1.79 ± 0.05   182 ± 32.7**   103 ± 12.4     71 ± 8.6      33 ± 6.6**	340 ± 23.4   1.80 ± 0.05   172 ± 45.1**   102 ± 22.6     71 ± 15.6      31 ± 13.3**

Pregnant rats were sacrificed on day 20 of gestation. Values are the mean ± S.D. of 8-11 animals per group. ^a(Body weight gain from day 0 to 20 of gestation) - (gravid uterus weight). *Significantly different from control, p < 0.05. **Significantly different from control, p < 0.01.

 

Conclusions:

Under the conditions of the study, the NOAEL for maternal toxictiy and foetal toxicity was determined to be 10 mg/kg bw/day.

Executive summary:

The maternal and fetal toxicity of dimethyltin chloride (DMTC) was examined in two teratological studies in pregnant Wistar rats. In one study, the animals were treated by oral gavage with DMTC at doses of 0, 5, 10, 15, or 20 mg/kg/day on gestational days 7-17. In the second study, animals were treated by oral gavage at doses of 0, 20 or 40 mg/kg/day DMTC on two or three consecutive days at one of four different periods of gestation (gestational days 7-9, 10-12, 13-15 or 16-17). Caesarean sections were performed on day 20 of gestation in both studies. In the first study, vaginal bleeding, tremor and convulsions were observed in animals treated at 20 mg/kg/day after day 15 of gestation. Of ten dams treated with 20 mg/kg/day DMTC, two died and one exhibited total resorption. While a increase in the incidence of cleft palate was found in the fetuses of animals treated with 20 mg/kg/day DMTC, the dams so treated exhibited severe clinical signs of toxicity. Animals treated in the second study with doses of 20 or 40 mg/kg/day at one of four periods of gestation had a reduction in the adjusted body weight gain but not in gravid uterus weight and did not show any evidence of teratogenic effect at either dose and period tested. These studies suggest that DMTC did not produce teratogenic effects at dose levels where no maternal toxicity was observed. It was suggested that under the conditions of the first study, since no signs of maternal or fetal toxicity could be detected up 10 mg/kg/day DMTC, this dose was chosen to represent the no-observed-adverse-effect level (NOAEL).

Reference 4

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across to structurally similar substance dimethyltin dichloride (DMTC) (EC Number 212-039-2, CAS Number 753-73-1), see attached justification.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

other: thymus atrophy

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

visceral malformations

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

2 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Two reliable key studies were available for this endpoint. The Noda (2001) was a more typical developmental study, similar to the OECD 414 guideline, whereas the Ehman (2007) paper focussed specifically on neurotoxicity. The overall quality of the database is therefore considered to be high.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Read-across to DMTC (Dimethyltin dichloride CAS No. 753 -73 -1)

Two developmental neurotoxicity studies (Ehman, 2007) were conducted with dimethyltin dichloride administered in drinking water at 3, 15 and 75 ppm. In the first study, female Sprague–Dawley rats were exposed 2 weeks prior to mating and throughout mating, gestation and lactation. Male offspring were compared to controls for differences in: 1) pre-weaning learning in an associative runway task, 2) motor activity ontogeny, 3) spatial learning and retention in the Morris water maze as adults, 4) brain weight, 5) biochemical evidence of apoptosis, and 6) neuropathology. Maternal toxicity was expressed as decreased maternal weight gain and decreased fluid intake. Only in the first of the two studies was developmental toxicity expressed as minimal to slight brain histopathology vacuolization of F1 pups/adults at all doses, decreased brain weights, and one of several neurobehavioral end points was altered.

In the second study, exposure to timed pregnant Sprague–Dawley rats occurred via drinking water from gestational day 6 to weaning and a similar battery of behavioural tests was administered to the F1 animals. The high concentration again depressed maternal weight gain and fluid intake. The F1 animals at the high dose had lower weight only during lactation, not thereafter. There was decreased brain weight but no correlating adverse histopathological findings in high-dose offspring.

Histopathological alterations of minimal to slight severity in the brain of offspring of dams exposed to DMT in Experiment 1 were noted in the cerebral cortex of rats sacrificed at PND22 and as adults. These lesions were not observed in Experiment 2 in any brain region. The foetal toxicity NOAEL was determined to be 15 ppm (equivalent to 2 mg/kg/day).

In the first teratology study (Noda, 2001), animals were treated on gestational days 7 -17. Vaginal bleeding, tremor and convulsions were observed in animals treated at 20 mg/kg/day after day 15 of gestation. Of ten dams treated with 20 mg/kg/day, two died and one exhibited total resorption. While an increase in the incidence of cleft palate was observed in fetuses of animals treated with 20 mg/kg/day DMTC, the dams so treated exhibited severe clinical signs of toxicity, including mortality, clinical signs of toxicity (piloerection, ataxia, perinasal and periocular staining, vaginal bleeding, tremor, and convulsion), and significantly reduced body weight gain and food consumption. Animals treated in the second study with doses of 20 or 40 mg/kg/day at one of four periods of gestation had a reduction in the adjusted body weight gain but not in gravid uterus weight and did not have an increase in the incidence of cleft palate or show any other evidence of a teratogenic effect at either dose or for any gestational period tested. These studies suggest that DMTC did not produce teratogenic effects at dose levels where no maternal toxicity was observed. As under the conditions of the first study no signs of maternal or fetal toxicity could be detected up 10 mg/kg/day DMTC, this dose was chosen to represent the NOAEL.

Toxicity to reproduction: other studies

Additional information

DMTC induced cleft palates on the fetuses at 20 mg/kg/day, in the presence of severe maternal toxicity at this high dose level (Noda 2001, first experiment). However, no significant increase in the incidence of cleft palates or other external, skeletal and visceral malformations were observed in a second study at similar or higher dose levels although the substance was administered for shorter durations but covering the whole embryogenesis period. Malformations were also not observed in Ehman 2007 but it may be due to lower dosage (high dose between 4 and 12 mg/kg).

DMTC induced a decrease in fetal body weight at 15 and 20 mg/kg (Noda 2001, first experiment). At these doses, maternal toxicity was also observed but the magnitude of fetal weight decrease (-17% and -37% in male pups and -15% and -34% in female pups) was more important that the magnitude of maternal weight decrease (-5% and -24%). No effect on fetal body weight was observed in a second study at similar or higher dose levels although the substance was administered for shorter durations and induced maternal toxicity as evidenced by significant decrease in maternal adjusted body weight gain. In Ehman 2007, a decrease in fetal body weight was observed only at high dose (7-12 mg/kg) in the second experiment during lactation when maternal weight was also significantly decreased. The link between foetotoxicity and maternal toxicity is therefore likely but cannot be totally excluded.

DMTC showed a developmental neurotoxic potential in Ehman 2007. The absence of reproductibility of the effects observed in the runaway and water maze tests does not provide a clear conclusion. Furthermore, the study is not consistent with guideline requirement, which raises further uncertainties on the significance of the result. However, it is noted that histopathological lesions observed only in experiment 1 are similar to those reported in adult exposed to DMTC and in foetuses exposed to MMTC, which support that this effect is specific to methyltin exposure and is treatment-related.

Due to the uncertainties discussed above, the evidence is not considered sufficient to place the substance in category 1B. A classification Repr. 2 H361d is proposed for DMTC.

Justification for classification or non-classification

In accordance with Annex VI of Regulation (EC) No. 1272/2008 (CLP) the substance should be classified Repr. 2 H361d