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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-04-21 to 2009-10-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
After dissolution and disociation in environmental (aqueous)/physiolological media the constituents of tin difluoride become bioavailable and the overall toxicity of the dissociated substance can be described by the toxicity of the individual constituents/ions. Since synergistic effects are not to be expected, the human health and environmental hazard assessment of the assessment entity tin difluoride consists of an individual assessment of the assessment entities tin cation and the fluoride anion. The tin cation and the fluoride anion are considered to represent the overall toxicity of tin difluoride in a manner proportionate to the fluoride and the metal (represented by one of its readily soluble salts). Based on the above information, unrestricted read-across is considered feasible and justified.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Version / remarks:
1983-05-26
Principles of method if other than guideline:
The execution of this study was in accordance with OECD 415 as applicable at the time of conduct.
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2007-11-09
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Tin(II)methane-sulfonate
- Molecular formula: Sn(CH3SO3)2

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS - Crl:WI(WU)
- Source: Charles River Deutschland, Germany
- Age at study initiation: approximately 6 weeks
- Housing: animals were housed individually in polycarbonate (Makrolon®) cages type III; absorbent softwood (Lignocel 3/4 or Lignocel BK 8-15) was used as bedding in the cages.
- Diet (ad libitum): closed formula commercial chow in pellet form identified as V1324 ( from ssniff Spezialdiaeten GmbH, Soest, Germany); Food was changed weekly or on the dates of determination of food consumption.
- Water (ad libitum): tap water was offered fresh weekly in 300 mL polycarbonate (Makrolon®) bottles
- Acclimation period: approximately 2 weeks

The daily observations showed that the animals were in good health and they were therefore accepted for this study.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 – 24 °C
- Relative humidity: 40 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet : once prior to study initiation, second as needed
- Mixing appropriate amounts with: V1324 (purchased from ssniff Spezialdiaeten GmbH, Soest, Germany)


Details on mating procedure:
- M/F ratio per cage: 1 male and 1 female of the same dose group
- Length of cohabitation: two consecutive weeks at maximum or until successful mating
- Proof of pregnancy: sperm and/or a vaginal plug was found; the day of finding sperm was considered day 0 post conception.
- Time to successful insemination (precoital time) was determined.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From each preparation and each concentration a sample was taken immediately after delivery for checking the content and confirm the identity of the test item. In addition, for confirming the stability of the test item preparation, from the first delivery of all three concentrations a second sample was analysed 5 weeks after delivery and a third sample 10 weeks after delivery.

The processing of the samples was as follows: After the sample (5 pellets) was powdered and homogenized, approx. 100 mg (exactly weighed) were suspended in 0.5 ml CD3CN/D2O (50/50 v/v). After addition of 10 μL of an aqueous solution of maleic acid (c=100 mg/mL) as internal standard the tin methylsulfonate was extracted ultrasonically. Quantification of the test item was then carried out by 1H NMR spectroscopy.

Results:
The analyses showed that preparations contained 97% (dose level: 1200 mg/kg), 104% (dose level: 3500 mg/kg), and 100 % (dose level: 7000 mg/kg) of the target concentration of the three dose groups.
Duration of treatment / exposure:
The animals were treated via food for 10 weeks before mating and during the mating period. After successful mating (day of finding sperm in vaginal smears = day 0 post conception [p.c.]), treatment of the females was continued until weaning of the offspring and subsequent sacrifice. Exposure of all males and females continued until their sacrifice after determination of sexual maturation in offspring. Animals selected for the investigation of sexual maturation were exposed from weaning to sacrifice.
The following observations/recordings of the parental animals were made during the study: clinical observations, body weight, food consumption, and gross pathology.
The following observations/recordings of the offspring were made during the study: clinical observations, number of offspring, occurrence of dead pups, abnormalities, pup weight, investigation of sexual maturation, organ weights, gross pathology and histopathology.
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg diet
Dose / conc.:
1 200 mg/kg diet
Remarks:
target dose: 100 mg SnMES/kg bw/day
Dose / conc.:
3 500 mg/kg diet
Remarks:
target dose: 300 and 600 mg SnMES/kg bw/day
Dose / conc.:
7 000 mg/kg diet
Remarks:
target dose: 600 mg SnMES/kg bw/day
No. of animals per sex per dose:
24 males / 24 females
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
The dose levels were based on the results of a 90-day study with 0, 50, 150, and 450 mg/kg bw per day administered by oral gavage. Mortality was observed in the high dose, and the NOEL was determined at 50 mg/kg/day.
Based on these results, a dose range finding study (Fraunhofer ITEM Study no. 12N08524) was performed based on OECD 421 "Reproduction/Developmental Toxicity Screening Test" using exposure via food at concentrations of 400, 1300, and 4000 mg/kg food (corresponding to actual doses of 35, 109, and 343 mg/kg/day in males and 38, 125, and 377 mg/kg/day in females, resp.). No effect on any of the investigated parameters was observed. In a second part of this DRF study, groups of 10 males (the more susceptible sex in the 90-day study) were exposed over 2 weeks to food exposure at concentrations of 4000, 5500, and 7000 mg/kg food (corresponding to actual doses of 375, 554, and 674 mg/kg/day, resp.). A marked reduction in body weight gain in the high dose group was observed in this study. Consequently, this dose was chosen as the high dose in the present study. The other doses were determined using a stagger of approx. 2-3, resulting in food concentrations of 7000, 3500, and 1200 mg/kg food in the present study.

Positive control:
none

Examinations

Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily; once per week, all animals were inspected outside their home cages.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly; after successful mating, body weight of the females was determined on days 0, 4, 7, 10, 14, and 20 post conception as well as 0, 4, 7, 14, and 21 post partum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Individual food consumption of the animals was recorded weekly by the weight difference between initial and remaining food. After successful mating, food consumption of the females was determined on days 0, 4, 7, 10, 14, and 20 post conception as well as 0, 4, 7, 14, and 21 post partum. The actual test item intake of the animals was calculated based on food consumption.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
- clinical observations: litters were inspected at least once daily.
- Survival and pup weight: number of offspring and the occurrence of any dead pups or abnormalities was recorded for all groups. Individual weight of the pups was recorded on days 0, 4, 7, 14, and 21 post partum.
- Postweaning investigations: after weaning of offspring on day 21 post partum, one male and one female per litter was selected randomly for postweaning investigation of sexual maturation. In these animals, the day of preputial separation and vaginal opening was determined and the body weight was recorded on the day of reaching the criterion. Selected offspring were sacrificed as soon as possible after reaching the criterion. Pups were weighed weekly until reaching sexual maturity.
All pups not selected for postweaning investigations were humanely sacrificed after weaning.

Postmortem examinations (parental animals):
SACRIFICE
After determination of sexual maturity in offspring, all F0 males and females were sacrificed by C02 overdose and subsequent exsanguination.

GROSS NECROPSY
Necropsy was performed in all F0 animals. The number of implantation sites was determined, using ammonium sulphide staining in case of no macroscopically visible implantations.
The following organs and tissues were collected from each rat and fixed in 10% neutral buffered formalin:
brain, pituitary, tongue, eyes, lacrimal glands, nasal and paranasal cavities, larynx, pharynx, trachea, thyroid, parathyroids, lungs, thymus, heart, aorta, lung-associated lymph nodes, salivary glands, mandibular lymph nodes, liver, pancreas, spleen, kidneys, adrenals, oesophagus, forestomach, glandular stomach, duodenum, jejunum, ileum, caecum, colon, rectum, mesenterium and lymph nodes, urinary bladder, testes (fixation in modified Davidson‘s fluid), epididymides (fixation in modified Davidson‘s fluid), prostate incl. coagulating glands, seminal vesicles, ovaries with oviduct (fixation in modified Davidson‘s fluid), uterus, vagina, mammary glands, skeletal muscle, femur including joint, vertebrae with spinal cord, skin, peripheral nerve, and sternum with bone marrow.

HISTOPATHOLOGY / ORGAN WEIGHTS
None
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 21 days of age except those evaluated for sexual maturation
- Necropsy was performed in all animals selected for investigations of sexual maturation. The following organs and tissues were collected from each rat and fixed in 10% neutral buffered formalin: brain, pituitary, tongue, eyes, lacrimal glands, nasal and paranasal cavities, larynx, pharynx, trachea, thyroid, parathyroids, lungs, thymus, heart, aorta, lung-associated lymph nodes, salivary glands, mandibular lymph nodes, liver, pancreas, spleen, kidneys, adrenals, oesophagus, forestomach, glandular stomach, duodenum, jejunum, ileum, caecum, colon, rectum, mesenterium and lymph nodes, urinary bladder, testes (fixation in modified Davidson‘s fluid), epididymides (fixation in modified Davidson‘s fluid), prostate incl. coagulating glands, seminal vesicles, ovaries with oviduct (fixation in modified Davidson‘s fluid), uterus, vagina, mammary glands, skeletal muscle, femur including joint, vertebrae with spinal cord, skin, peripheral nerve, and sternum with bone marrow.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The organ weights of uterus, ovaries, testes, epididymides, seminal vesicles, and prostate were determined.
The following organs and tissues from each rat of the high-dose and control group were investigated histopathologically: vagina, uterus, oviducts, cervix, ovaries, testes, epididymides, seminal vesicles, prostate, and coagulating glands.
Statistics:
Statistical comparison of groups was performed separately for each sex at the level of α =0.05. Parental and offspring weights as well as food consumption were analysed using analysis of variance. If the group means differed significantly with this method, the means of the treatment groups were compared with the mean of the control group 1 using Dunnett's modification of the t-test. Kruskall-Wallis ANOVA and Mann-Whitney U-test were applied in the case of non-normal data.
Qualitative reproduction data were analysed using the two-tailed FISHER test with Bonferroni correction or Chi-square test. In the results the p-value indicates the minimum α for which the Bonferroni-corrected test was significant. Histopathology data were analysed using the two-tailed FISHER test.
In all instances, the dam or litter was used as the basic unit. Postmating data and organ weights of females without implantation sites were excluded from statistical analysis. All statistical analyses were run using Toxicology Analysis System Customized, version 0, or SAS, version 6.12, on an alpha microcomputer.
Reproductive indices:
Female fertility index, male fertility index, female mating index, male mating index, gestation index
Offspring viability indices:
Livebirth index, viability index, lactation index

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All animals of the medium and high dose group showed yellow-brown discoloured faces throughout the study, obviously as a consequence of substance excretion via faeces. These were not considered adverse effects.
Other clinical signs like red exsudate around snout, conjunctivitis, lacrimation, exophthalmos, ulceration or alopecia were occasionally observed in single animals from all groups including the control group. None of these findings were considered test item related.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There was a significant decrease in both body weight and body weight gain at the start of treatment in medium and high dose males as well as in high dose females.
In males, the differences in body weight from the control group reached statistical significance in the high and medium dose group on days 7-28 and 42, in the high dose group additionally on day 56, 84 and 140. Body weight gain in males was statistically significantly decreased dose-dependently between day 0 and 7 in the medium and high dose group, with values returning to normal afterwards. Sporadical statistically significant increases or decreases in body weight gain observed after day 84 in all groups compared to the control group are considered incidental.
In females, body weight was statistically significantly reduced in the high dose group only on days 7, 14 and 28, while body weight gain was reduced in the high dose group only between days 0 and 7. The statistically significant increase in body weight gain observed between day 14 and 21 in medium dose group females is considered incidental.
No effects were observed on gestation body weight, gestation body weight gain and maternal postpartum body weights. However, postpartum weight gain was statistically significantly increased in the high dose group from day 14 p.p. onwards and also for the total period, as well as in the medium dose group on day 21. These effects certainly cannot be considered adverse, but led to a catch-up of body weights in females towards the end of the lactation period.
There was a significant decrease in food consumption at the start of treatment in medium and high dose males as well as in high dose females.
In males, the differences from the control group reached statistical significance in the high and medium dose group between day 0 and 7. The same was observed for high dose females only. These effects are in good concord with the influences observed on body weight gain and can be interpreted as a repellent effect of the food containing higher doses of the test item.
All values returned to normal afterwards, sporadical statistically significant increases or decreases observed after day 7 in all groups compared to the control group are considered incidental.
No effects on food consumption were observed on food consumption during gestation or lactation.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Based on mean body weights and food consumption data, this resulted in the following actual substance intake:
- 73, 225, and 454 mg/kg body weight/day for males;
- 97, 303, and 605 mg/kg body weight/day for females in the premating period;
- 80, 259, and 522 mg/kg body weight/day for females during gestation;
- 215, 634, and 1297 mg/kg body weight/day for females during lactation

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
For the four groups, mating yielded 24, 22, 23, and 22 sperm positive females, respectively. This resulted in 23, 19, 22, and 21 pregnancies and 23, 19, 22, and 21 females with liveborn offspring, respectively. One animal (low dose group), no sperm could be found in vaginal smear, but the animal gave birth.
Based on these results, no effect of test item treatment on mating and pregnancy rates as well as on precoital time could be observed in this study.
No effect was observed on the number of implantation sites, number of live pups or pup loss during lactation, or duration of gestation.

GROSS PATHOLOGY (PARENTAL ANIMALS)
None of the sporadically occurring necropsy observations was considered test item induced. Consequently, no effect was observed on necropsy observations in F0 animals.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
> 7 000 other: mg SnMES/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: target dose: 600 mg SnMES/kg bw/day (228 Sn/kg bw/day), no effects on fertility and reproductive performance of parental animals were observed.
Remarks on result:
other:
Remarks:
The authors themselves conservatively proposed NOELS for SnMES of 3500 mg/kg food (target dose of 300 mg/kg body weight/day; tin: 114 mg/kg bw/day)) for developmental endpoints, and 1200 mg/kg food (target dose of 100 mg/kg body weight/day; tin: 38 mg/kg bw/day) for parental endpoints. However, these values were based on observed effects only at the start of treatment in the parental generation, and in selected postweaning offspring high dose males and high and medium dose females. In preweaning offspring, the only effect observed was a decrease of pup weight in the second half of lactation, which reached statistical significance in the high dose only group on day 21. It is important to consider that this effect occurred only when pups began to eat food in addition to maternal lactation, and therefore can be interpreted as a repellent effect of the food containing higher doses of the test item, which was also observed in the beginning of treatment in F0 animals and not as a specific reproductive or developmental effect, which can also be the reason for the observed slight effects on offspring gonads. Thus, the no adverse effect level in this one-generation reproduction toxicity study is established at a dose level of 7000 mg/kg in the diet (target dose of 600 mg/kg).

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

CLINICAL SIGNS (OFFSPRING):
Clinical findings in pups consisted mainly of poor condition observed in single pups of all groups. The only remarkable finding was unilateral anophthalmia in one single pup of one dam (high dose).

BODY WEIGHT (OFFSPRING)
No effect was observed on pup birth weight
The only effect observed was a decrease in pup weight in the second half of lactation, which reached statistical significance in the high dose group on day 21 (day of weaning). This effect occurred only when pups began to eat food in addition to maternal lactation and, therefore, can be interpreted as a repellent effect of the food containing higher doses of the test item, which was also observed in the beginning of treatment in F0 animals.
There was a trend towards a decreased pup body weight in all test item treated groups, which reached statistical significance in males of the high dose group, and in females of the medium and high dose group. In males, body weight gain remained decreased during the whole post-weaning observation period, while in females the only statistically significant decrease was observed between day 28 and 35 in the medium dose group.
These effects, again, can be interpreted as a repellent effect of the food containing higher doses of the test item, which was also observed in the beginning of treatment in F0 animals.

SEXUAL MATURATION (OFFSPRING):
No effect was observed on the time of showing preputial separation or vaginal opening in any of the test item treated groups. There was, however, a trend towards a decrease in body weight on the day of completion of sexual maturity, which reached statistical significance in males of the high dose group. This can be interpreted as a consequence of the reduced body weight observed in pups of this dose group at weaning as well as later on.

ORGAN WEIGHTS (OFFSPRING):
The only statistically significant differences on organ weights were observed in the high dose group for absolute weights of testes, epididymides and ovaries. However, no differences were observed for any relative organ weights in any of the test item exposed groups.

GROSS PATHOLOGY (OFFSPRING):
None of the sporadically occurring necropsy observations was considered test item induced.
Consequently, no effect was observed on necropsy observations in F1 animals.

HISTOPATHOLOGY (OFFSPRING)
- Testes: slight (multi)focal tubular atrophy was seen in one out of 23 males of the control and 2/21 males of the high dose group. Very slight/slight and moderate degeneration of round spermatids and/or pachytene spermatocytes was detected in four males of the high dose group and associated with slight depletion of round and elongated spermatids in one of these males. Degeneration of spermatids was also observed in 1/23 control males at a very slight severity.
- Epididymides: moderate hypospermia and exfoliation of germ cells into the efferent ducts was found in two males of the high dose group (2/21). Two males (2/21) of the high dose group showed only very slight exfoliation of germ cells into the epididymides. (Multi)focal very slight interstitial mononuclear cell infiltration was seen in 6/23 males of the control group and in 7/21 males of the high dose group. One male (1/23) of the control group revealed a small granuloma.
- Prostate: very slight interstitial/intra-acinar inflammatory cell infiltration was observed in 4/23 males of the control and in 1/21 males of the high dose group.
- Uterus: very slight or slight dilated uterine horns were observed in 5/23 females of the control group and in 7/21 females of the high dose group. These dilatations are due to accumulation of fluid dependent on hormonal influences during the oestrous cycle.

OTHER FINDINGS (OFFSPRING) No effect was observed on sex ratio of pups.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 7 000 other: mg SnMES/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: target dose: 600 mg SnMES/kg bw/day (228 Sn/kg bw/day), no specific effects on pre-and post-natal development of offspring were observed.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The no observed adverse effect level of tin(II)methane-sulfonate in this One-generation reproduction toxicity study in Wistar WU rats was determined
at a concentration of 3500 mg/kg food (target dose of 300 mg/kg body weight/day; tin: 114 mg/kg bw/day)) for developmental endpoints, and 1200 mg/kg food (target dose of 100 mg/kg body weight/day; tin: 38 mg/kg bw/day) for adult endpoints.
These values are based on the finding that body weight and body weight gain at the start of treatment of the parental generation were decreased in males of the high and medium dose as well as females of the high dose group. This effect was also replicated in selected postweaning offspring high dose males and high and medium dose females. In preweaning offspring, the only effect observed was a decrease of pup weight in the second half of lactation, which reached statistical significance in the high dose only group on day 21. This effect occurred only when pups began to eat food in addition to maternal lactation and, therefore, can be interpreted as a repellent effect of the food containing higher doses of the test item, which was also observed in the beginning of treatment in F0 animals and not as a specific reproductive or developmental effect, and can also be the reason for the observed slight effects on offspring gonads.
Thus, in this one-generation reproduction toxicity study, no effects on fertility and reproductive performance of parental animals and no specific effects on pre-and post-natal development of offspring were observed up to a dose level of 7000 mg/kg in the diet (target dose of 600 mg/kg).