Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 October 2009 and 16 December 2009 (the in life-phase of the study was performed between 06 October 2009 (first day of treatment) and 30 November 2009 (final necropsy)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
6 October 2009 and 16 December 2009 (the in life-phase of the study was performed between 06 October 2009 (first day of treatment) and 30 November 2009 (final necropsy)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Blackthorn, Bicester, Oxon U.K.
- Age at study initiation: circa 12 weeks
- Weight at study initiation: 335 to 381 g (males) and 185 to 234 g (females)
- Fasting period before study: No
- Housing: Initially in groups of five by sex in solid floored polypropylene cages with softwood bedding. During mating, one male and one female were co-housed in polypropylene grid floor cages suspended over trays lined with absorbent paper. Following evidence of mating the males were returned to their original cages and females were housed individually through gestation and lactation in solid floored polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet: ad libitum access to pelleted diet - Rodent 2018C Teklad Global Certifed Diet, Harlan Laboratories, Oxon, UK.
- Water: ad libitum access to mains potable water via polycarbonate cage bottles
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.80 - 23.02 °C
- Humidity: 45.88 - 71.94 %
- Air changes: at least 15 per hr
- Photoperiod: 12/12 (hrs dark / hrs light; low intensity fluorescent lighting)


IN-LIFE DATES: From: 5 October 2009 To: 16 December 2009
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Tin metal pwder was prepared at the appropriate concentrations as a suspension in 1 % (w/v) carboxy methylcellulose (sodium salt). The stability and homogeneity of the test material formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services (Harlan Laboratories Ltd. Project Number: 2712-0005 - see summary of 28-day repeated dose toxicity IUCLID point 7.5.1). Results showed the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly during the treatment period and stored at approximately 4 ºC in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The choice of vehicle was selected based on information from previously performed toxicity studies.
- Concentration in vehicle: 10, 30 or 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Details of the analytical verification of doses were referenced in the Harlan Laboratories Ltd report 2712-0005

Due to the nature of the test material and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis was not developed. The concentration of Tin Metal Powder (2-11 µm) in the test material formulations was determined using a gravimetric technique.

SAMPLES
The test material formulations were weighed into tared glass beakers and then dried in an oven overnight at approximately 105 °C before allowing to cool over silica gel in a desiccators and re-weighed.

HOMOGENEITY
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate. On one occasion samples were left at ~105 °C over three days and not overnight, this was considered not to impact on the scientific integrity of the study.

STABILITY DETERMINATIONS
The test material formulations were sampled and analysed initially and then after storage at approximately + 4 °C in the dark for fourteen days.

VERIFICATION OF TEST MATERIAL FORMULATION CONCENTRATIONS
The test material formulations were sampled and analysed within three days of preparation.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days (max.) from Day 15, pre-coitus interval was typically 4 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear - referred to as day 0 of pregnancy (Gestation Day 0)
- After successful mating each pregnant female was caged (how): individually in solid floor polypropylene cage through gestation
- Any other deviations from standard protocol: No
Each pregnant female was observed three times daily (approximately 0830, 1230 and 1630 hours) (or twice on weekends and holidays) and around the period of expected parturition. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

Vaginal smears were prepared and oestrus staging determined throughout pair housing
Duration of treatment / exposure:
56 days
Frequency of treatment:
Daily
Duration of test:
Chronological Sequence of Study
i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iii) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
iv) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and surface righting reflex were also performed during this period.
v) The male dose groups were killed and examined macroscopically on Day 43.
vi) At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Ten males and ten females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses selected on basis of previous toxicity investigations (Harlan Study number 2712-0005, see section 7.5.1) Limit dose based on absence of findings in a repeated oral subacute rat study.
- Rationale for animal assignment (if not random): random
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one and five hours after dosing during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends (except during parturition where applicable). All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to dosing) then at weekly intervals to termination until mating was noted. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
During the maturation period, weekly food consumption was recorded for each cage of adults until pairing. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4).
Weekly food efficiency was calculated retrospectively during the pre-mating period and during the first two weeks of gestation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily visual assessment of overt changes in water bottles only - no gravimetric or volumetric assessments

SACRIFICE
- Maternal animals: All surviving female dams were killed on day 5 post-partum. All adults were subject to external and internal examination. Dams were also examined for uterine signs of implantation and number of implantation sites per horn counted.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
Samples of the following tissues were preserved for histopathological examination: ovaries, mammary tissue, uterus/cervix, vagina and pituitary

The tissues from control and 1000 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
- pre and post-implantation loss indices: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: No
- Head examinations: No
Statistics:
Statistical Analyses are presented under section "Any other information on materials and methods"
Indices:
- Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

- Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated/Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females/Number of animals mated) x 100

- Gestation and Parturition Data
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation:
i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring/Number of pregnant females) x 100

- Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation losses were calculated for each female/litter as follows:
% pre-implantation loss =[ (Number of corpora lutea - number of implantation sites)/Number of corpora lutea] x 100
% post-implantation loss = [(Number of implantation sites – Total number of offspring born)/Number of implantation sites] x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1/Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4/Number of offspring alive on Day 1) x 100
iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following:
Sex Ratio (% males) = (Total number of offspring/Number of male offspring) x 100
Historical control data:
Normal ranges were provided for the following parameters:
Normal Ranges for Reproductive Parameters in the Pregnant and Lactating Female Wistar HanTM:HsdRCCHanTM: WIST Strain Rat
Normal Ranges for Offspring Parameters in the Wistar HanTM:HsdRCCHanTM: WIST Strain Rat
Normal Ranges for Absolute and Bodyweight Relative Organ Weight Values in the Wistar HanTM:HsdRCCHanTM: WIST Strain Rat.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One female treated with 1000 mg/kg/day showed noisy respiration on Day 11 only. In the absence of any associated changes and due to the presence of the observation also in a control animal the intergroup differences were considered not to be toxicologically significant. One control had generalised fur loss between Days 14 and 45 and chromodacryorrhea between Days 32 and 34. One female treated with 100 mg/kg/day also had generalised scab formation between Days 40 and 42. Observations of this nature are commonly observed in studies of this type and in isolation are considered not to be toxicologically significant.
Mortality:
no mortality observed
Description (incidence):
No mortalities occurred in the parental generation.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No adverse effect on bodyweight development was detected. Statistical analysis did not reveal any significant intergroup differences.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effects on food consumption were detected during the course of the study.
Food efficiency:
no effects observed
Description (incidence and severity):
No adverse effects on food efficiency were detected during the course of the study.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No adverse effects on water consumption were detected during the course of the study.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Statistical analyses showed no significant intergroup variations. No effect on organ weights was detected.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No toxicologically significant effects apparent. The only changes noted - fur loss and scab formation were not considered to be toxicologically significant
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related findings observed.
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Group mean implantation counts and implantation losses indicated no effect of maternal exposure.
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
Group mean corpora lutea, litter size and survival indices all indicated no effect of maternal exposure on offspring viability.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Gestation length was not significantly affected and no effects on parturition were recorded. All females gave birth within 22 to 23.5 days.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): There were no significant intergroup differences in gestation lengths and no effects upon parturition for treated females, with all of the females giving birth following 22 to 23.5 days of gestation. The distribution for treated females was comparable to controls.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were no effects on conception rates.
Other effects:
no effects observed
Description (incidence and severity):
There were no exposure related effects on mating performance. The distribution of precoital intervals for treated animals was comparable to controls with the majority of animals showing positive evidence of mating within four days of pairing (i.e. the first oestrus opportunity). Evidence of mating was generally apparent within 4 days of pairing (first oestrous opportunity).
Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No toxicologically significant effects observed at the limit dose level in this screening study
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Total litter weights and individual weights and individual bodyweight gains were unaffected by maternal exposure.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Post natal survival was unaffected in all treated groups
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter size at Day 1 for treated animals was similar to controls.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Post natal survival was unaffected in all treated groups with litter size at Day 4 being similar to controls.
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No signs of any effect of treatment. The type, incidence and distribution of clinical signs and percentage of offspring successfully showing a surface righting reflex was unaffected by maternal exposure from day 1 post-partum to termination on day 5.
No macroscopic effects attributable to treatment were apparent.
Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically significant effects observed at the limit dose level in this screening study
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no

Tabular Summary Report of Effects on Reproduction/Development

Observations

Dose Level (mg/kg/day)

0 (Control)

100

300

1000

Mated pairs

n

10

10

10

10

Females showing evidence of copulation

n

10

10

10

10

Pregnant females

n

10

10

10

10

Conception Days 1-14

n

10

10

10

10

Gestation = 22 Days

n

1

1

2

2

Gestation = 22 ½ Days

n

6

1

2

6

Gestation = 23 Days

n

3

3

4

1

Gestation = 23 ½ Days

n

0

5

2

1

Dams with live young born

n

10

10

10

10

Dams with live young at Day 4post partum

n

10

10

10

10

Corpora lutea/dam  

x

15.2

13.8

15.9

17.2

Implants/dam

x

12.9

12.8

13.1

14.9

Live offspring/dam Day 1post partum

x

11.6

11.8

12.8

14.0

Live offspring/dam at Day 4post partum

x

11.4

11.6

12.5

13.7

Sex ratio: % males Day 1post partum

x

53.8

47.8

48.9

44.0

Sex ratio: % males at Day 4post partum

x

54.0

47.0

49.6

44.1

Litter weight (g) at Day 1post partum

x

69.2

66.8

69.6

73.0

Litter weight (g) at Day 4post partum

x

100.8

98.1

99.8

102.2

Male offspring weight (g) at Day 1post partum

x

6.1

5.9

5.7

5.4

Male offspring weight (g) at Day 4post partum

x

9.0

8.8

8.4

7.7

Female offspring weight (g) at Day 1post partum

x

5.8

5.6

5.4

5.1

Female offspring weight (g) at Day 4post partum

x

8.7

8.4

8.0

7.4

LOSS OF OFFSPRING/DAM

 

 

 

 

 

Pre-implantation (corpora lutea minus implantations)

 

 

 

 

 

0

n

4

7

5

4

1

n

0

0

1

1

2

n

0

0

0

1

3

n

1

0

1

0

5

n

1

0

0

0

6

n

1

0

1

0

7

n

1

0

0

2

18

n

0

0

1

0

Pre-natal (implantations minus live births)

 

 

 

 

 

0

n

3

3

7

1

1

n

1

1

3

4

2

n

4

3

0

2

4

n

0

1

0

0

5

n

0

0

0

1

Post natal (live births minus offspring alive on Day 4post partum)

 

 

 

 

 

0

n

8

8

8

8

1

n

2

2

1

1

2

n

0

0

1

1

n = Number, x  = Mean

Conclusions:
The oral adminsitration of tin metal powder (2-11 µm) to rats by gavage, for a period of up to fifty-six consecutive dats at dose levels of 100, 300 and 1000 mg/kg/day did not result in any treatment-related effects.
The No Observed Effect Level (NOEL) was therefore considered to be 1000 mg/kg/day.
Executive summary:

The study was performed to screen for potential adverse effects of tin metal powder on reproduction and offspring development and provides an initial hazard assessment for effects on reproduction. The study was compliant with OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (1995) andCommission Regulation (EC) No 440/2008 testing requirements.

Tin metal powder (2 -11 µm) was administered by gavage to three groups, each of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, for up to fifty-six consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (1 % (w/v) aqueous carboxy methylcellulose (sodium salt)).

Clinical signs, bodyweight development, dietary intake and water consumption were monitored during the study. 

The parental rats were paired one to one from day 15 of treatment and females were allowed to litter and rear young to day 5 post-partum. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Adult males were terminated on Day 43, and all females and surviving offspring on Day 5post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

There were no deaths and no significant clinical signs of toxicity. No effects on parental bodyweight or weight gain were noted, nor were there any effects on food consumption. The reproductive parameters assessed in this screening study showed no changes or adverse effects attributable to exposure to tin metal powder.

The litter response also showed no effects of maternal exposure that could be attributed to tin metal powder (2 -11 µm) toxicity.

The offspring raised to day 5 post-partum showed no effects from maternal exposure to metallic tin. Developmental landmarks assessed in the study showed the offspring were unaffected by treatment. The study shows metallic tin administered orally at up to 1000 mg/kg bw/day for 56 days to rats has no toxicologically significant effect on reproductive and developmental parameters.

No biologically significant effects were apparent at the 1000 mg/kg dose level.

The data was therefore considered sufficient for classification and labelling purposes.

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 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): tin metal powder (2-11 µm )
- Substance type: metal powder ground to ensure particle size was within the range of 2-11 µm
- Physical state: grey metallic powder
- Lot/batch No.: 090418
- Storage condition of test material: room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Blackthorn, Bicester, Oxon U.K.
- Age at study initiation: circa 12 weeks
- Weight at study initiation: 335 to 381 g (males) and 185 to 234 g (females)
- Fasting period before study: No
- Housing: Initially in groups of five by sex in solid floored polypropylene cages with softwood bedding. During mating, one male and one female were co-housed in polypropylene grid floor cages suspended over trays lined with absorbent paper. Following evidence of mating the males were returned to their original cages and females were housed individually through gestation and lactation in solid floored polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet: ad libitum access to pelleted diet - Rodent 2018C Teklad Global Certifed Diet, Harlan Laboratories, Oxon, UK.
- Water: ad libitum access to mains potable water via polycarbonate cage bottles
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.80 - 23.02 °C
- Humidity: 45.88 - 71.94 %
- Air changes: at least 15 per hr
- Photoperiod: 12/12 (hrs dark / hrs light; low intensity fluorescent lighting)

IN-LIFE DATES: From: 5 October 2009 To: 16 December 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Tin metal pwder was prepared at the appropriate concentrations as a suspension in 1 % (w/v) carboxy methylcellulose (sodium salt). The stability and homogeneity of the test material formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services (Harlan Laboratories Ltd. Project Number: 2712-0005 - see summary of 28-day repeated dose toxicity IUCLID point 7.5.1). Results showed the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly during the treatment period and stored at approximately 4 °C in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The choice of vehicle was selected based on information from previously performed toxicity studies.
- Concentration in vehicle: 10, 30 or 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days (max.) from Day 15, pre-coitus interval was typically 4 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear - referred to as day 0 of pregnancy (Gestation Day 0)
- After successful mating each pregnant female was caged (how): individually in solid floor polypropylene cage through gestation
- Any other deviations from standard protocol: No
Each pregnant female was observed three times daily (approximately 0830, 1230 and 1630 hours) (or twice on weekends and holidays) and around the period of expected parturition. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Details of the analytical verification of doses were referenced in the Harlan Laboratories Ltd report 2712-0005

Due to the nature of the test material and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis was not developed. The concentration of Tin Metal Powder (2-11 µm) in the test material formulations was determined using a gravimetric technique.

SAMPLES
The test material formulations were weighed into tared glass beakers and then dried in an oven overnight at approximately 105 °C before allowing to cool over silica gel in a desiccators and re-weighed.

HOMOGENEITY
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate. On one occasion samples were left at ~105 °C over three days and not overnight, this was considered not to impact on the scientific integrity of the study.

STABILITY DETERMINATIONS
The test material formulations were sampled and analysed initially and then after storage at approximately + 4 °C in the dark for fourteen days.

VERIFICATION OF TEST MATERIAL FORMULATION CONCENTRATIONS
The test material formulations were sampled and analysed within three days of preparation.
Duration of treatment / exposure:
56 days
Frequency of treatment:
Daily
Details on study schedule:
Chronological Sequence of Study
i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iii) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
iv) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and surface righting reflex were also performed during this period.
v) The male dose groups were killed and examined macroscopically on Day 43.
vi) At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.

Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Ten males and ten females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses selected on basis of previous toxicity investigations (Harlan Study number 2712-0005, see section 7.5.1) Limit dose based on absence of findings in a repeated oral subacute rat study.
- Rationale for animal assignment (if not random): random
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one and five hours after dosing during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends (except for females during parturition where applicable). All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to dosing) then at weekly intervals to termination for males and at weekly intervals until mating was noted for females. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
During the maturation period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4).
Weekly food efficiency was calculated retrospectively for males and for females during the pre-mating period and during the first two weeks of gestation.

WATER CONSUMPTION : Yes
- Time schedule for examinations: Daily visual assessment of overt changes in water bottles only - no gravimetric or volumetric assessments
Oestrous cyclicity (parental animals):
Vaginal smears were prepared and oestrus staging determined throughout pair housing
Sperm parameters (parental animals):
No data
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: not applicable - study terminated on Day 5 post-partum

PARAMETERS EXAMINED - Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum by placing the offspring on its back on a flat surface and observing for its ability to turn over to its normal resting position within a one minute period.

On completion of parturition (Day 0 post-partum) the numbers of live and dead offspring were recorded. For each litter the following data were also recorded:
number of offspring born
number of offspring alive recorded daily and reported on Days 1 and 4 post partum
offspring were sexed on days 1 and 4 post-partum
clinical condition checked daily from birth to day 5 post-partum
individual offspring weights and litter weights recorded on days 1 and 4 post-partum
Surviving offspring were terminated on day 5 post-partum
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving males were killed on Day 43
- Maternal animals: All surviving female dams were killed on day 5 post-partum. All adults subject to external and internal examination . Dams were also examined for uterine signs of implantation and number of implantation sites per horn counted.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Epididymides and testes from the terminal kill males were weighed

HISTOPATHOLOGY / ORGAN WEIGHTS
Samples of the following tissues were preserved for histopathological examination:
males only: coagulation gland, prostate, epididymides, seminal vesicles, testes
females only: ovaries, mammary tissue, uterus/cervix, vagina
both sexes: pituitary

The tissues from control and 1000 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. In addition sections of testes and epididymides from all control and 1000 mg/kg/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Postmortem examinations (offspring):
GROSS EXAMINATION OF DEAD PUPS:
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.
Surviving offspring and those dying during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
Statistical Analyses are presented under section "Any other information on materials and methods"
Reproductive indices:
- Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

- Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated/Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females/Number of animals mated) x 100

- Gestation and Parturition Data
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation:
i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring/Number of pregnant females) x 100
Offspring viability indices:
- Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation losses were calculated for each female/litter as follows:
% pre-implantation loss =[ (Number of corpora lutea - number of implantation sites)/Number of corpora lutea] x 100
% post-implantation loss = [(Number of implantation sites – Total number of offspring born)/Number of implantation sites] x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1/Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4/Number of offspring alive on Day 1) x 100
iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following:
Sex Ratio (% males) = (Total number of offspring/Number of male offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One female treated with 1000 mg/kg/day showed noisy respiration on Day 11 only. One control male also showed noisy respiration on Day 40. In the absence of any associated changes and due to the presence of the observation also in a control animal the intergroup differences were considered not to be toxicologically significant. One control had generalised fur loss between Days 14 and 45 and chromodacryorrhea between Days 32 and 34. One female treated with 100 mg/kg/day also had generalised scab formation between Days 40 and 42. Observations of this nature are commonly observed in studies of this type and in isolation are considered not to be toxicologically significant.
Mortality:
no mortality observed
Description (incidence):
No mortalities occurred in the parental generation.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Males - No toxicologically significant effect on bodyweight development was detected. Males treated with 1000 mg/kg/day showed a statistically significant increase in bodyweight gain during Week 3. An increase in bodyweight gain is not considered to represent an adverse health effect therefore the intergroup difference is considered not to be toxicologically important.
Females - No adverse effect on bodyweight development was detected. Statistical analysis did not reveal any significant intergroup differences.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effects on food consumption were detected during the course of the study.
Food efficiency:
no effects observed
Description (incidence and severity):
No adverse effects on food efficiency were detected during the course of the study.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No adverse effects on water consumption were detected during the course of the study.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related findings observed.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no exposure related effects on mating performance. The distribution of pre-coital intervals for treated animals was comparable to controls with the majority of animals showing positive evidence of mating within four days of pairing (i.e. the first oestrus opportunity). Evidence of mating was generally apparent within 4 days of pairing (first oestrous opportunity). There were no effects on conception rates. Gestation length was not significantly affected and no effects on parturition were recorded. All females gave birth within 22 to 23.5 days.

FERTILITY
There were no treatment-related effects detected in conception rates. Group mean corpora lutea, implantation counts and implantation losses all indicated no effect of maternal exposure.

GESTATION LENGTH
There were no significant intergroup differences in gestation lengths and no effects upon parturition for treated females, with all of the females giving birth following 22 to 23.5 days of gestation. The distribution for treated females was comparable to controls.

Effect levels (P0)

Key result
Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects at highest dose tested
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No effect of maternal exposure on the clinical behaviour of the offspring.
No signs of any effect of treatment. The type, incidence and distribution of clinical signs and percentage of offspring successfully showing a surface righting reflex was unaffected by maternal exposure from day 1 post-partum to termination on day 5.
Mortality / viability:
no mortality observed
Description (incidence and severity):
No treatment related effects apparent.
Group mean litter size and survival indices all indicated no effect of maternal exposure on offspring viability at dose levels of 100, 300 or 1000 mg/kg /day. Subsequent litter size at Day 1 for treated animals was similar to controls. Post natal survival was unaffected in all treated groups with litter size at Day 4 again being similar to controls.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Total litter weights and individual weights and individual bodyweight gains were unaffected by maternal exposure.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No evidence of any effect of maternal exposure on offspring macroscopic appearance.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects at highest dose tested
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Tabular Summary Report of Effects on Reproduction/Development

Observations

Dose Level (mg/kg/day)

0 (Control)

100

300

1000

Mated pairs

n

10

10

10

10

Females showing evidence of copulation

n

10

10

10

10

Pregnant females

n

10

10

10

10

Conception Days 1-14

n

10

10

10

10

Gestation = 22 Days

n

1

1

2

2

Gestation = 22 ½ Days

n

6

1

2

6

Gestation = 23 Days

n

3

3

4

1

Gestation = 23 ½ Days

n

0

5

2

1

Dams with live young born

n

10

10

10

10

Dams with live young at Day 4post partum

n

10

10

10

10

Corpora lutea/dam  

x

15.2

13.8

15.9

17.2

Implants/dam

x

12.9

12.8

13.1

14.9

Live offspring/dam Day 1post partum

x

11.6

11.8

12.8

14.0

Live offspring/dam at Day 4post partum

x

11.4

11.6

12.5

13.7

Sex ratio: % males Day 1post partum

x

53.8

47.8

48.9

44.0

Sex ratio: % males at Day 4post partum

x

54.0

47.0

49.6

44.1

Litter weight (g) at Day 1post partum

x

69.2

66.8

69.6

73.0

Litter weight (g) at Day 4post partum

x

100.8

98.1

99.8

102.2

Male offspring weight (g) at Day 1post partum

x

6.1

5.9

5.7

5.4

Male offspring weight (g) at Day 4post partum

x

9.0

8.8

8.4

7.7

Female offspring weight (g) at Day 1post partum

x

5.8

5.6

5.4

5.1

Female offspring weight (g) at Day 4post partum

x

8.7

8.4

8.0

7.4

LOSS OF OFFSPRING/DAM

 

 

 

 

 

Pre-implantation (corpora lutea minus implantations)

 

 

 

 

 

0

n

4

7

5

4

1

n

0

0

1

1

2

n

0

0

0

1

3

n

1

0

1

0

5

n

1

0

0

0

6

n

1

0

1

0

7

n

1

0

0

2

18

n

0

0

1

0

Pre-natal (implantations minus live births)

 

 

 

 

 

0

n

3

3

7

1

1

n

1

1

3

4

2

n

4

3

0

2

4

n

0

1

0

0

5

n

0

0

0

1

Post natal (live births minus offspring alive on Day 4post partum)

 

 

 

 

 

0

n

8

8

8

8

1

n

2

2

1

1

2

n

0

0

1

1

n= Number, x  = Mean

Applicant's summary and conclusion

Conclusions:
The oral adminsitration of tin metal powder (2-11 µm) to rats by gavage, for a period of up to fifty-six consecutive days at dose levels of 100, 300 and 1000 mg/kg/day did not result in any treatment-related reproductive effects.
The No Observed Effect Level (NOEL) for reproductive toxicity was therefore considered to be 1000 mg/kg/day.
Executive summary:

The study was performed to screen for potential adverse effects of tin metal powder on reproduction and offspring development and provides an initial hazard assessment for effects on reproduction. The study was compliant with OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (1995) and Commission Regulation (EC) No 440/2008 testing requirements.

Tin metal powder (2 -11 µm) was administered by gavage to three groups, each of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, for up to fifty-six consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (1 % (w/v) aqueous carboxy methylcellulose (sodium salt)).

Clinical signs, bodyweight development, dietary intake and water consumption were monitored during the study.  The parental rats were paired one to one from day 15 of treatment and females were allowed to litter and rear young to day 5 post-partum.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Adult males were terminated on Day 43, and all females and surviving offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

There were no deaths and no significant clinical signs of toxicity. No effects on parental bodyweight or weight gain were noted, nor were there any effects on food consumption. The reproductive parameters assessed in this screening study showed no changes or adverse effects attributable to exposure to tin metal powder.

The litter response also showed no effects of maternal exposure that could be attributed to tin metal powder (2 -11 µm) toxicity.

The reproduction/fertility and developmental no observed effect level in rats was greater than the limit dose of 1000 mg/kg bw/day. No biologically significant effects were apparent at this dose level.

The data was therefore considered sufficient for classification and labelling purposes.