Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a GLP compliant reproductive/developmental toxicity screening study conducted in accordance with guideline OECD 421 the oral administration 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.

Link to relevant study records
Reference
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
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
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
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.

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
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
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: 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.
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
Key result
Critical effects observed:
no
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
Key result
Reproductive 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 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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The quality of the database is considered to be high.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Dunster J (2010) was performed in compliance with GLP and in accordance with the OECD guideline 421, the study was assigned a reliability score of 1 in accordance with the criteria outlined in Klimisch et al. (1997). The study was considered adequate and reliable for assessment, and sufficient for classification and labelling purposes. No higher tier testing is proposed.

Effects on developmental toxicity

Description of key information

SCREENING STUDY

In a GLP compliant reproductive/developmental toxicity screening study conducted in accordance with guideline OECD 421 the oral administration 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 developmental effects was therefore considered to be 1000 mg/kg/day.

PRENATAL DEVELOPMENTAL TOXICITY STUDY

In a GLP compliant prenatal developmental toxicity study conducted in accordance with guidelines OECD 414, EU Method B.31, US EPA OPPTS 870.3700 and JMAFF, the oral administration of tin metal powder (2-11 µm) to rats by gavage during Days 6 to 20 of gestation at dose levels of 30, 300 and 1000 mg/kg/day did not result in any treatment-related reproductive or developmental effects.

The No Observed Adverse Effect Level (NOAEL) for developmental effects was therefore considered to be 1000 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:WI(Han)
- Age at study initiation: Females were nine to ten weeks old
- Animal receipt: Females were delivered by Day 3 of gestation
- Weight at study initiation: Females weighed at least 140 g at mating. Females weighed between 167.3 and 252.9 g at the start of dosing.
- Fasting period before study: No
- Housing: The animals were housed singly in cages with Aspen wood chips used for bedding (changed on a weekly basis). Animals were provided with wooden Aspen chew blocks and rodent nesting materials as forms of environmental enrichment.
- Diet: Food was provided ad libitum throughout the study
- Water: Mains water was provided ad libitum via water bottles
- Acclimation period: Acclimatisation was limited by mated status

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24 °C
- Humidity: 45 to 65 % (relative)
- Air changes: 15 to 20 per hour
- Photoperiod: 12 hrs dark / 12 hrs fluorescent light

IN-LIFE DATES
From: 03 August 2015
To: 21 August 2015
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.1 % CMC (sodium salt)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
- Frequency of preparation: Formulations were prepared weekly
- Storage of dosing solutions: The formulations were stored refrigerated (2 to 8 °C) in a sealed container
- Test material administration: On arrival at the animal room, the formulations were stirred continuously for thirty minutes before and throughout dosing (excluding the vehicle control group).

VEHICLE
- Amount of vehicle (if gavage): A dose volume of 10 mL/kg was used. Individual dose volumes were based on individual body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
STABILITY AND HOMOGENEITY
- Stability: Determined at 3 and 100 mg/mL for 14 days at 4 °C in the dark in a previous study
- Homogeneity: Determined at 3 and 100 mg/mL in a previous study

CONCENTRATION VERIFICATION
Samples were taken from formulations prepared for the first and last day of dosing. Three samples (1 mL) removed from the test material formulations were added to pre-weighed HPLC vials. These samples were analysed to determine the achieved concentration. Two samples (1 mL) were removed from the control formulations to determine the vehicle subtraction weight.
Details on mating procedure:
- Impregnation procedure: Overnight at the supplier’s laboratory
- Proof of pregnancy: Presence of a vaginal plug in situ, or other evidence of mating, if necessary. The day on which mating was confirmed was designated as Day 0 of gestation.
- Other: Females were considered to be sexually mature at the start of dosing.
Duration of treatment / exposure:
From Day 6 to 20 of gestation
Frequency of treatment:
Once daily
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:
20 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high dose level of 1000 mg/kg/day was the No-Observed-Effect-Level (NOEL) in the rat OECD 421 screening study. In the event of treatment related effects, 300 mg/kg/day was considered to be an appropriate step between the low and high dose levels to help characterise findings. The low dose level, 30 mg/kg/day was expected to be a NOEL with respect to adult toxicity and embryo-foetal development.
- Rationale for animal assignment: The animals were assigned to treatment groups on arrival based on body weight and day of mating (i.e. all animals confirmed as mated on a specific day were randomly allocated to treatment groups).
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at the beginning and the end of the working day for signs of ill health or overt toxicity. The animals were observed at approximately 1 hour after dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination on the days of body weight recording. An individual record was maintained of the clinical condition of each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on Day 3, 6, 7, 8, 9, 12, 15, 17, 19, 20 and 21 of gestation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: The amount of food consumed was determined daily beginning on Day 3 of gestation. Food consumption was calculated as g/animal/day.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21. Females were euthanised in replicate order on Day 21 of gestation by cervical dislocation following isoflurane anaesthesia, death confirmed by exsanguination. Food was not removed prior to necropsy. All animals examined macroscopically. All lesions were recorded and retained in the relevant fixative.
- Organs examined: Ovaries and uteri
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: Yes
- Number of late resorptions: Yes
- Other: The number and intrauterine position of implantations was subdivided into live foetuses, early intrauterine deaths, late intrauterine deaths and dead foetuses.
Early intrauterine deaths were classified as those which showed decidua or placental tissue only. Late intrauterine deaths showed embryonic or foetal tissue in addition to placental tissue. Dead foetuses were classified as those which appeared to have died shortly before necropsy. Implantations were allocated numbers relating to their position in utero.
The uterus of any apparently non pregnant female was immersed in a 10 % ammonium sulphide solution to reveal any evidence of implantation.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No

Live foetuses were euthanised by a subcutaneous injection of sodium pentobarbitone.
Individual foetal and placental weights were recorded and foetuses were examined externally and sexed. Approximately one half of the foetuses in each litter, selected by systematic sampling, were dissected and the visceral abnormalities were examined. They were then eviscerated and the carcasses processed for skeletal examination. The skeletons were examined in 50 % glycerol and retained in glycerol/propylene glycol.
The remaining foetuses were fixed in Bouin's fluid and examined. The foetuses fixed for visceral examination were retained in the relevant fixative.
Foetal abnormalities were classified as malformations (rare and/or potentially lethal defects) and variations (commonly occurring non-lethal abnormalities).
Statistics:
Data from treated animals were compared with control data. Statistical analyses were performed where appropriate.
Data for each sex was analysed separately, unless stated otherwise. Except where otherwise stated, tests were performed using a two-sided risk and were considered significant where P≤0.05.
Body weight, body weight gains and food consumption were analysed using analysis of variance (ANOVA). Mean foetal weight (male, female and combined) was analysed using analysis of covariance (ANCOVA), with litter size (live and dead foetuses) as the covariate. The number of corpora lutea, implantation sites, early and late resorptions, dead foetuses, live foetuses and percent pre- and post-implantation loss and percent male foetuses were analysed using the Kruskal-Wallis and Wilcoxon rank sum test.
Gravid uterine weights and corrected body weights (carcass weights) were analysed using analysis of variance (ANOVA) and the percentage of foetuses affected (mean litter percent) was analysed using the Kruskal-Wallis and Wilcoxon rank sum test.
Proportion of litters affected was analysed using a 1–sided upper tail Fishers exact test.
Full details are given below.
Indices:
A number of indices were used, where appropriate, to evaluate reproductive function:
- % pre-implantation loss = [(Number of corpora lutea – number of implantations) / Number of corpora lutea] x 100
- % post-implantation loss = [(Number of implantations – number of live embryos) / Number of implantations] x 100
- Carcass weight = Terminal body weight – uterus weight
- Total weight change = Day 21 body weight – Day 3 body weight
- % male foetuses = (Number of male foetuses / Number of foetuses of determined sex) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical observations were consistent across groups and unaffected by treatment. Observations consisted primarily of fur and/or coat changes which are common background observations in the rat. There were no post-dosing observations associated with treatment.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight parameters were unaffected by the test material at all dose levels.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by the test material at all dose levels.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic observations associated with treatment. Caesarean section parameters were consistent across all dose groups and unaffected by treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
DOSE ANALYSIS
The target range for the preparation of the formulations was 90 to 100 % of nominal. All results were within this range and considered acceptable for use.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Reproductive performance was consistent across all groups and unaffected by treatment.
Details on maternal toxic effects:
Sixteen females with implantations per group are considered optimal for statistical analysis of data. In this study there were fifteen females with implantations in the control group. Since the study results are clear, and statistical analysis is not needed to discern the lack of effects on pregnancy and embryo-foetal development, this is not considered to have adversely affected interpretation of the study.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
Foetal external parameters were consistent across all groups and unaffected by treatment.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Foetal skeletal parameters were consistent across all groups and unaffected by treatment.
Visceral malformations:
no effects observed
Description (incidence and severity):
Foetal visceral parameters were consistent across all groups and unaffected by treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1: Summary of Reproductive Performance

Treatment Group

Control

30 mg/kg

300 mg/kg

1000 mg/kg

Total Females

20

20

20

20

Pregnant Females

15

17

17

19

Non Pregnant Females

5

3

3

1

Pregnant with Total Litter Loss

1

1

1

0

Females with Live Foeuses

14

16

16

19

Table 2: Summary of Mean Caesarean Section Data

Treatment Group

Control

30 mg/kg

300 mg/kg

1000 mg/kg

Number of females pregnant at caesarean section

15

17

17

19

Corpora lutea

12.6

12.9

11.8

12.5

Implantation sites

10.2

11.2

10.9

11.2

Pre-implantation loss

2.4

1.7

0.9

1.3

Pre-implantation loss (%)

18.8

11.5

8.3

11.6

Early resorptions

0.5

0.4

0.6

0.5

Late resorptions

0.0

0.0

0.0

0.0

Total Resorptions

0.5

0.4

0.6

0.5

Dead foetuses

0.0

0.0

0.0

0.0

Post-implantation loss

0.5

0.4

0.6

0.5

Post-implantation loss (%)

11.5

8.7

10.0

4.1

Live foetuses

10.4†

11.5†

10.9†

10.8

†The number of females examined was reduced due to excluded data

 

Table 3: Summary of Mean Foetal Data

Treatment Group

Control

30 mg/kg

300 mg/kg

1000 mg/kg

Number of females with live foetuses

14†

16†

16†

19

Mean number of male foetuses per litter

4.6†

6.2†

5.8†

5.5

Mean number of female foetuses per litter

5.8†

5.3†

5.2†

5.3

% Male foetuses

48.5†

54.1†

53.0†

48.1

Mean foetal weight (g)

5.34†

5.31†

5.41†

5.41

Mean¹ foetal weight - males (g)

5.41†

5.41†

5.51†

5.49

Mean¹ foetal weight - females (g)

5.14†

5.28†

5.28†

5.23

†The number of females examined was reduced due to excluded data

Conclusions:
Under the conditions of this study, daily administration of the test material at doses up to and including 1000 mg/kg/day had no effect on parameters evaluated within this study and the No Observed Adverse Effect Level (NOAEL) is therefore concluded to be 1000 mg/kg/day for both maternal toxicity and developmental toxicity.
Executive summary:

A pre-natal developmental toxicity study was carried out in the rat to investigate the effects of the test material on embryonic and foetal development. The study was conducted in accordance with the standardised guidelines OECD 414, EU Method B.31, US EPA OPPTS 870.3700 and JMAFF under GLP conditions.

Pregnant female Wistar rats were exposed to the test material in 0.1 % carboxymethylcellulose at dose levels of 0, 30, 300 and 1000 mg/kg/day (20 per group) on Days 6 to 20 of gestation. The females were maintained to Day 21 of gestation when they were sacrificed and their uterine contents examined.

Assessment of toxicity was based on clinical signs, body weight and food consumption. Complete necropsies were performed on all animals with a recording of macroscopic abnormalities for all tissues. Caesarean and foetal examinations were conducted.

There were no unscheduled deaths in the course of the study. Clinical observations, body weight parameters and food consumption were consistent across all groups and unaffected by treatment at doses up to 1000 mg/kg/day.

There were no macroscopic observations associated with treatment and reproductive performance was consistent across all groups and unaffected by treatment. Caesarean section parameters were consistent across all dose groups and unaffected by treatment

Foetal external, visceral and skeletal parameters were consistent across all groups and unaffected by treatment at doses up to 1000 mg/kg/day.

Under the conditions of this study, daily administration of the test material at doses up to and including 1000 mg/kg/day had no effect on parameters evaluated within this study and the No Observed Adverse Effect Level (NOAEL) is therefore concluded to be 1000 mg/kg/day for both maternal toxicity and developmental toxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The quality of the database is considered to be high as there are two good quality studies available; a screening study and a prenatal developmental toxicity study both performed to standardised guidelines under GLP conditions.
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

The supporting screening study, Dunster J (2010), was performed in compliance with GLP and in accordance with the OECD guideline 421; the study was assigned a reliability score of 1 in accordance with the criteria outlined in Klimisch et al. (1997).

 

The prenatal developmental toxicity study, Rhodes J (2016), was performed in compliance with GLP and in accordance with OECD 414, EU Method B.31, US EPA OPPTS 870.3700 and JMAFF; the study was assigned a reliability score of 1 in accordance with the criteria outlined in Klimisch et al. (1997).

Toxicity to reproduction: other studies

Additional information

In accordance with Column 2 (8.7 Reproductive toxicity) of Annex X of Regulation (EC) No. 1907/2006, reproductive toxicity studies need not be conducted if the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and there is no or no significant human exposure.

There was no evidence of any toxicological activity of metallic tin in any endpoint measured, up to the limit dose (1000 mg/kg bw/day) in any of the following studies: the fully compliant OECD 407 28 day repeat dose Rat study (McRae, 2010, NOAEL >1000mg/kg bw/day based on the absence of any adverse effects), the fully compliant OECD 421 reproductive screening study (Dunster, 2010, NOAEL for reproductive, foetal and parental endpoints measured was >1000mg/kg bw/day, based on the absence of any adverse effects), the fully compliant OECD 414 developmental toxicity study (Rhodes, 2016, NOAEL for developmental, foetal and maternal endpoints measured was >1000mg/kg bw/day, based on the absence of any adverse effects). It was not acutely toxic via any route, was not irritating or sensitising to skin. As metallic tin is an inorganic heavy metal there is little scope for species differences in terms of developmental toxicity, with mechanisms of heavy metal toxicity usually pertaining to non-specific (and non-species specific) denaturing of proteins, and homeostatis of other important cations, e.g. iron, copper, zinc (see e.g TOXNET http://toxnet.nlm.nih.gov/) HSDB entry for TIN CASRN: 7440-31-5). Hence the the absence of these non-specific chemical effects in one species are sufficient to infer absence of effects in the second species species. The UK competent authority also constructed a similar although slightly different case why developmental toxicity studies were not required for tin. See ECHA Communication number CCH-D-0000004928-60-02/D attached.

Exposure assessment also indicates there is very limited human exposure, despite wide and dispersive use. In workers the highest anticipated inhalation exposure without PPE or engineering control is typically 4-5 mg/m3, peaking at 10 mg/m3, whilst dermally it is upto 9.9 mg/day. Engineering controls or PPE will be required to reduce this to comply with non-specific nuisance dust rules (the OEL for non-specific nuisance dust is frequently specified as 10 mg/m3 total inhalable and/or with a respirable fraction no greater than 3 mg/m3), hence these exposures will be considerably lower. There is no anticipated consumer exposure where tin is in solid metallic form either pure or in an alloy.

It can be seen from the toxicokinetic data (which utilised extreme exposures), when put into the context of normal human background dietary exposure and plasma concentrations, that metalic tin would not be absorbed by the oral route to any detectable degree under normal occupational exposure conditions (see the toxicokinetics, metabolism and distribution section i.e. absorption for risk assessment purposes can be considered zero). The additional pathology conducted in the acute inhalation toxicity study (Gaunt, 2009) also showed that there would be no practical systemic absorption of metallic tin, via the inhalation or for that matter dermal routes of exposure.

Hence further reproductive toxicity testing, or a second species developmental toxity studies need not be conducted:

In light of the studies conducted so far, and the absence of any practical level of absorption, it is clear that no further useful information would be gleaned from such studies and would be inappropriate in terms of animal welfare.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to reproductive or developmental toxicity (teratogenicity) as the available data indicates that there is no cause for concern.