Tin, dioctylbis(2,4-pentanedionato-.kappa.O2,.kappa.O4)-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No ewffect, maternal effects << reproductive effects, se dissussion

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12 November 2003 to 8 March 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted in accordance with the standardised testing guideline OECD 422 and in accordance with GLP with no deviations thought to affect the quality of the presented data. The study was reported to a high standard, sufficient to assess the reliability of the data presented.

Justification for type of information:

Tin, dioctylbis(2,4-pentanedionato-κO2,κO4)- / hydrolyse in contact with humidity / water in the stomach into Dioctyloxastannanne and 2,4-Pentadione. This endpoint is the assessment for Dioctyloxastannanne

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10-11 weeks
- Weight at study initiation: The weight variation of the animals for each sex did not exceed 20 %.
- Diet: ad libitum
- Water: tap water ad libitum in polypropylene bottles

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 30-70 5
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

DOSE-RANGE FINDING TEST IN-LIFE DATES: From: 12 November 2003 To: 26 November 2003
MAIN TEST IN-LIFE DATES: From: 14 January 2004 To: 8 March 2004

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): The experimental diets were prepared once shortly before the study
- Mixing appropriate amounts with (Type of food): The test substance was weighed and placed in a small grinder. The tray was rinsed with food which was then also added to the grinder and mixed for 2 x 30 seconds. This mixture was moved to a Stephan cutter and the grinder was rinsed with food and moved to the cutter. Approximately 3 kg weighed food was mixed into the cutter for 2 x 2 minutes and moved. This was then moved to the Lödige cutter. The Stephan cutter was rinsed with approximately 3 kg of food and that was also moved to the Lödige cutter. Mixing was continued in the Lödige cutter for 2 minutes with the total amount of food.
- Storage temperature of food: <-18 °C

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Until pregnancy occurred
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The feed was checked for homogenous distribution, stability and concentration of the test substance for all the doses in the dose-range finding study. The same dose preparation for the dose-range finding study was also used for the main test. The homogenous distribution and achieved concentration of the low dose in the main test was also analysed.
Directly after preparation of the diet for the dose-range finding study, samples for homogeneity and stability were taken. Five samples were taken (approximately 50 g each) to examine the homogeneity of the dose from the top centre, middle centre, bottom centre, left centre and right centre of the mixer. Secondly samples (around 50 g) were taken from the top centre part of the mixer to measure the stability. The samples taken for measurement of the homogeneity were also used for dose confirmation. In addition the content (achieved concentration) of the test substance in the batch of diet used in the main study. Diet samples were taken for analysis immediately after preparation and stored at – 18 °C.
Samples of the 0, 25, 75, 200 and 500 mg/kg diets from the dose-range finding study and doses of 0, 25 and 250 mg/kg from the main study as well as all related calibration samples were derivatised.
A calculated amount of internal standard solution (MHT, DHT and TTPT in methanol) was added to 2.0 g of diet in a 50 mL Corning tube. 10 mL of 100 % acetic acid was then added and the Corning tube was closed and shaken for 60 minutes (250 rpm). 10 mL of acetate buffer solution (pH 4.5) was added along with 10 mL methanol. 2.0 mL of 20 % (m/V) aqueous STEB solutions was added, followed by 10 mL hexane (containing naphthalene, approximately 0.1 mg/L). The tube was shaken for 15 minutes (250 rpm) then placed in an oven at 60 °C for 15 minutes. After phase separation, the hexane layer (approximately 3 mL) was removed and washed with 3 mL of 2 mol/L HCl (30 minutes shaking at 250 rpm). The hexane top layer was diluted with hexane: hexane extracts from sample with dose levels of 0, 25 and 75 mg/kg were diluted five times, the higher doses were diluted fifty times. The resulting solutions were transferred into an amber coloured glass vial and anaylysed using GC-MS.
The procedure for the samples from the main study at doses of 0 and 5 mg/kg followed the same derivatisation procedure, except the initial quantity of the diet was 5.0 g not 2.0 g
The concentration of organotin compounds in the extracts was determined using GC-MS. For calculation of the amount of DOTO in the samples, the peak area of DHT was used as an internal standard. Quantitiation was achieved using the calibration graphs constructed from the calibration solutions.
The following conditions were used:
- Column: Fused silica HP5 MS, 30 m, 0.25 mm ID, 0.25 µm film
- Precolumn: fused silica HP5 MS, 2.5 m, 0.25 mm ID, 0.25 µm film
- Column temperature: after 3 minutes at 45 °C at a rate of 5 °C/min to 80 °C; then at a rate of 15 °C/min to 260 °C; 15 min at 260 °C.
- Carrier: helium; 1.5 mL/min constant flow
- Injection volume: 1 µL
- Injection temperature: start at 60 °C, then at a rate of 14.5 °C/s to 300 °C; 5 min at 300 °C
- Injection method: splitless
- Ionisation: electron impact 70 eV
- Mass range: 60-600 amu
- Mass fragments used: DOT m/z = 375*; 263; 151, DHT 347*; 249; 179
Mass fragments marked with an asterisk were used for quantitation

Duration of treatment / exposure:

28 days (treated food was available ad libitum)

Frequency of treatment:

Daily

Details on study schedule:

Doses of the range finder:
Doses / Concentrations:
Range finding study: 0, 25, 75, 200 and 500 mg/kg diet
Basis:
nominal in diet

Dose / conc.:

0 mg/kg diet

Remarks:

Doses / Concentrations:
Main study: 0, 5, 25 and 250 mg/kg diet
Basis:
nominal in diet

Dose / conc.:

5 mg/kg diet

Dose / conc.:

25 mg/kg diet

Dose / conc.:

250 mg/kg diet

No. of animals per sex per dose:

22 males and 22 females in the 14-day dose range finding study (5 groups of 4 male and 4 female rats)
52 males and 52 females in the main study (4 groups of 12 male and 12 female rats)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Doses were selected on the results of the range finding test
- Rationale for animal assignment: Randomised

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Every morning throughout the study, and a second observation in the afternoon of working days.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the first exposure and then once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of male and female rats were taken on day -2 (randomisation) and on days 0 (first day of dosing), 7 and 13 of the premating period.
Males were weighed weekly during the mating period until sacrifice. Females were weighed during mating (day 0, 7 and 13) and mated females were weighed on day 0, 7, 14 and 21 during presumed gestation and on day 1 and 4 of lactation. All animals were weighed at sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, also calculated as g/animal/day

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the premating period
- Anaesthetic used for blood collection: Yes, CO2/02 anaesthesia
- Anit-coagulant: K2-EDTA
- Animals fasted: Yes, overnight
- How many animals: 5 rats/sex/group
- Parameters checked:
Haemoglobin
Packed cell volume
red blood cell count
reticulocytes
Total white blood cell count
Prothrombin time
Thrombocyte count
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the premating period
- Anaesthetic used for blood collection: Yes, CO2/02 anaesthesia
- Animals fasted: Yes, overnight
- How many animals: 5 rats/sex/group
- Parameters checked:
Fasting glucose
Alkaline phosphatase activity (ALP)
Aspartate aminotransferace activity (ASAT)
Alanine aminotransferace activity (ALAT)
Gamma glutamyl transferase activity (GGT)
Total protein
Albumin
Ratio albumin to globulin
Urea
Creatinine
Bilirubin (total)
Cholesterol (total)
Triglycerides
Phospholipids
Calcium (Ca)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Inorganic phosphate

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Arena testing was performed prior to the first exposure and then once weekly until the end of dosing and in females until the end of lactation. Males and females selected for the functional observation battery test (FOB) and spontaneous activity measurements were excluded from the final arena testing.
At the end of the study, FOB test and spontaneous motor activity measurements were performed on day 27 for males and on post natal day for for females.
- Dose groups that were examined: All animals were subject to the arena testing. For the other two tests, 5 animals per sex were randomly selected from each dose group.
- Battery of functions tested:
Autonomic: lacrimation, salivation, pupil response to light, palpebral closure, piloerection, defecation and urination
Neuromuscular: gait, mobility, forelimb and hindlimb gripstrength, landing foot splay, righting reflex
Sensorimotor: response to tail pinch, click, tough and approach of a visual object
Convulsive: clonic and tonic movements
Excitability: ease of removal, handling reactivity, arousal and vocalisations
Activity: rearing and motor activity
Physiological: body temperature

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in the offspring:
The total litter size and numbers of each sex as well as the number of stillbirths, livebirths and grossly malformed pups and pups showing abnormalities were recorded on PN 1 and PN 4. The pups were weighed individually on PN 1 and PN 4. Mean pup weights were calculated as litter weight/number pups. The number of runts (pup weight less than mean pup weight of the control group minus 2 standard deviations) was calculated.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Males were sacrificed after 28 days of exposure
- Female animals: Sperm positive females that were not pregnant were killed 25 days after copulation, mothers with litters were killed on post natal day 4.

GROSS PATHOLOGY: Yes
The following were taken from all animals:
Ovaries
Uterus
Testes
Epididymides
Seminal vesicles
Prostate
Organs and tissues showing macroscopic abnormalities
The following were taken from 5 animals/sex/group:
Adrenals
Axillary lymph node
Bone marrow (femur)
Brain
Caecum
Coagulation glands
Colon
Duodenum
Eyes
Heart
Jejunum
Lungs
Kidneys
Liver
Mammary gland (females only)
Mesentric lymph node
Parathyroids
Peyer's patches
Pituitary
Rectum
Sciatic nerve
Spinal cord
Spleen
Stomach
Thymus
Thyroids
Trachea
Urinary bladder

The following organs were weighed:
Adrenals
Brain
Heart
Kidneys
Liver
Spleen
Thymus

HISTOPATHOLOGY: Yes Microscopic examination was performed on the collected organs of all rats in the control and high-dose group.
The liver and ovaria of females and the thymus of the male and female rats in the low and mid-dose groups were also evaluated
The following tissues, though collected were not subject to histopathological examination:
Coagulation glands
Mammary gland (females only)
In addition, reproductive organs of males that failed to sire (mated female which was not pregnant) and females that were non-mated or non-pregnant of the mid and low dose groups were microscopically examined.

Postmortem examinations (offspring):

SACRIFICE
- The offspring were sacrificed at post natal day 4 by hypothermia at <-18 °C.
Pups were examined externally for gross abnormalities.

GROSS NECROPSY
- Pups that died during the study were necropsied and macroscopic abnormalities were recorded.

Statistics:

Clinical findings, histopathological changes, the number of mated and pregnant females and females with live pups were evaluated using Fisher’s exact probability test.
Number of implantation sites, live and dead pups were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann Whitney U-test.
Bodyweight, bodyweight gain, organ weights, food consumption, red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values and organ weights were assessed by one-way ANOVA followed by Dunnett’s multiple comparison tests.
Reticulocytes and relative differential white blood cell counts were assessed using Kruskal-Wallis non-parametric ANOVA followed by Mann-Whitney U-tests.
The results of the functional observations were measured on different scales. Continuous measurements were analysed by one-way analysis of variance at each time point, if found to be statistically significant, a post-hoc group comparison was performed. Rank order data were analysed by Kruskal-Wallis analysis of variance at each test time point, followed by planned multiple comparisons between dose groups were a significant results occurred. Categorical data were assessed using Pearson chi-square analysis.
Motor activity data were assessed by one-way analysis of variance at each time point with a post-hoc group comparison performed on significant results.
All tests were two sided and the level of probability p<0.05 was considered as significant. Effects of treatment on habituation were analysed by repeated measures of analysis variance in five 6 minute time blocks. Statistical evaluations on pup variables were considered on a litter basis. Additional evaluations on a pup basis were performed to identify any specific dose-related effect that may have occurred.

Reproductive indices:

Pre-coital time = time between the start of mating and successful copulation
Duration of gestation = time between gestation day 0 and day of delivery
Mating index = (number of females mated/number of females placed with males) x 100
Male fertility index = (number of males that became sires/number of males placed with females) x 100
Female fertility index = (number of pregnant females/number of females placed with males) x 100
Female fecundity index = (number of pregnant females/number of females mated) x 100
Gestation index = (number of females with live pups/number of females pregnant) x 100

Offspring viability indices:

Live birth index = (number of pups born alive/number of pups born) x 100
Pup mortality day n = (Number of dead pups/number of females pregnant) x 100
Live birth index = (number of pups born alive/number of pups born) x 100
Pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
Sex ratio day n = (number of live male pups on day n/number of live pups on day n) x 100
Number of lost implantations = number of implantation sites – number of pups born alive
Pre-implantation loss = [(number of corpora lutea – number of implantation sites)/number of corpora lyutea] x 100
Post-implantation loss = [(number of implantation sites – number of pups born alive)/number of implantation sites] x 100

Clinical signs:

effects observed, treatment-related

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY
One female in the high dose group was found dead on gestation day 24; this animal was pregnant and 11 dead foetuses were found in the uterus.
One male animal in the high dose group showed exopthalmus from week 2 and complete degeneration of the eye from week 3 onwards. This was observed after orbital punction. No other clinical signs were noted in the males.
The only finding during the gestation period was a sparsely haired animal in the 250 mg/kg group. During the lactation period, sparsely haired animals were noted in the control (n = 1), 5 mg/kg (n = 1) and in the 250 mg/kg group (n = 1). No other findings were noted in the female animals.

BODY WEIGHT AND WEIGHT GAIN
Mean bodyweights of the treated male animals was comparable to the controls. The bodyweight change of the male animals of the high-dose group was significantly decreased from days 13-21 (body weight gain 69% lower than controls).
Mean bodyweights of the dams of the high-dose group was statistically significantly decreased on gestation day 21 (8.5% lower than controls) and post-natal day 1 (9.3% lower than controls). Mean bodyweight changes of the dams of the high-dose group were more markedly affected during pregnancy, as the overall mean weight gain for the control animals accounted to 74.31 g, whereas that in the high-dose animals it was only 51.43 g (approx. 31% lower); durig gestation day 14 to 21, the reduction in mean weight gain in the high-dose animals attained statistical significance, compared to controls, and was approximately 52% lower. Bodyweights and bodyweight change of the dams of all the treated groups were comparable to the controls group at all other times of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Mean food consumption (g/kg/day) of male animals of the 250 mg/kg diet group was decreased by 7.5%, compared to controls, from day 7-13, and the difference attained statistical significance. No other treatment related effects were observed in the male animals.
During the gestation period and lactation period (gestation days 7-14 and 14-21 and post-natal days 1-4), food consumption (expressed as g/animal/day and g/kg bodyweight/day) of the dams in the high-dose group was significantly decreased. Considering the entire pregnancy period, high-dose animals ate approx. 13% less food than controls (in terms of g/kg/day), and approx. 24% less food than controls during the 4-day lactation period. No other treatment-related effects were observed.

Test substance intake was as follows:
- Males (listed as low, mid and high dose groups; 5, 25 and 250 mg/kg diet respectively)
premating days 0-7: 0.4, 1.7 and 17.4 mg/kg bodyweight/day
premating days 7-13: 0.3, 1.6 and 15.4 mg/kg bodyweight/day
post-mating days 21-28: 0.3, 1.5 and 14.5 mg/kg bodyweight/day

- Females (listed as low, mid and high dose groups; 5, 25 and 250 mg/kg diet respectively)
premating days 0-7: 0.4, 1.7 and 17.4 mg/kg bodyweight/day
premating days 7-13: 0.3, 1.6 and 15.4 mg/kg bodyweight/day
gestation days 0-7: 0.4, 2.0 and 17.4 mg/kg bodyweight/day
gestation days 7-14: 0.4, 1.9 and 16.7 mg/kg bodyweight/day
gestation days 14-21: 0.3, 1.4 and 11.2 mg/kg bodyweight/day
Post-natal days 1-4: 0.5, 2.4 and 17.4 mg/kg bodyweight/day

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The number of pregnant females and the number of males that became sires were: 11, 12, 11 and 10 for the control, low, mid and high dose groups respectively. The number of females with liveborn pups was 11, 11, 11 and 8 for the control, low, mid and high dose respectively.
The mating index was 100% in all groups. The female fecundity index, female fertility index and male fertility index were comparable among the all groups and ranged from 83-100%. The gestation index was comparable 100% in the control, low- and mid-dose groups, and 80% in the high-dose group.
The duration of gestation was statistically significantly increased in the 250 mg/kg diet (high-dose) group, although all animals gave birth between day 21 and 22. One female was found dead in the high dose group on gestation day 24. The animal was pregnant and 11 dead foetuses were discovered in the uterus. No difference on the duration of gestation was observed amongst the control and the low- and mid-dose groups.
Stillborn pups were observed in the high dose in three litters, and 1 female with all stillborn pups was observed.
Pre-implantation loss was 8.4. 2.2. 12.3 and 6.2% for the control, low-, mid- and high-dose groups and was considered comparable.
The statistically increased number of implantation sites in the 5 mg/kg diet (low-dose) group was not considered to be treatment related.
Pos-implantation loss was 9.8, 11.6, 7.5 and 38.7% for the control, low-, mid- and high-dose groups respectively. The post-implantation loss recorded in the high-dose group was statistically significantly increased.

HAEMATOLOGY
All treated groups were found to be comparable to controls.

CLINICAL CHEMISTRY
Statistically significantly increased alkaline phosphatase levels (U/L) were found in the high-dose males. Bilirubin (µmol/L) was found to be statistically significantly increased in the high-dose females. These findings were considered to be treatment related. Other effects such as the statistically significant increase in chloride in the 25 mg/kg diet male rats and the significant decrease in calcium in the 5 mg/kg diet females were not considered to be related to treatment. No other changes were observed.

NEUROBEHAVIOUR
No treatment-related effects were observed.

ORGAN WEIGHTS
The absolute thymus weight in the males of the 250 and 25 mg/kg diet groups were found to be significantly decreased (-23 and -80%, respectively, compared to controls). Relative thymus weight was found to be significantly decreased in the high dose male rats (-77% compared to controls).
The absolute and relative weight of the female animals of the high dose group was significantly decreased (-69 and -66%, respectively, compared to controls). In the mid-dose group the relative thymus weight was also statistically significantly decreased (-36% compared to controls).
In the female animals of the high-dose group, the relative kidney and liver weights were statistically significantly increased (+14 and +22%, respectively, compared to controls).
No other effects were observed in either male or female animals.

GROSS PATHOLOGY
At necropsy, a decrease in thymic size was seen in all animals in the 250 mg/kg diet groups, 11 females in the 25 mg/kg diet group, 7 females in the 5 mg/kg diet group and 5 animals in the control group.
Examination of the female that was found dead revealed hydrothorax, haemorrhagic lungs, dilation of the vena cava and haemorrhagic discharge in the vagina, these were considered to be indicative of problems during parturition.

HISTOPATHOLOGY
Microscopic evaluation of the thymus revealed moderate to very severe lymphoid depletion in all animals (both sexes) of the 250 mg/kg diet group and in all females of the 25 mg/kg diet group. Lymphoid depletion was characterised by a decrease in the thymic lobules due to an extensive loss of cortical and medullary small lymphocytes. The distinction between the cortical and medullary areas was unclear. In the more extreme effects observed, the cortex was very small, or absent. The remaining lymphoid cells visible in the cortical areas were mainly lymphoblasts. Lymphoblastic cells and reticuloepithelial cells had increased, and/or higher numbers of these cells were visible due to the disappearance of small lymphocytes and the collapse of the thymic stroma. In 3 high-dose females, lymphoid depletion was accompanied by lymphoid depletion in the PALS (periateriolar lymphocyte sheath areas) in the spleen. The macroscopically observed thymi in 5 control and 7 low dose females exhibited no microscopic abnormalities. In the thymi of the 2 control and 2 low dose females pregnancy/lactation involution was observed. The thymic lobules were decreased in size but exhibited normal structure with the histological appearance of age-involution. Increased glycomeric vacuolation, viz moderate versus very slight was seen in the liver of 4 high dose females and was considered to be a potential cause of the increased weight.
Examination of the reproductive organs revealed a statistically significant increased in the incidence of cysts in the ovaries of 8 high-dose females.

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

> 0.3 - < 0.4 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Decreased thymus weight

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

> 0.3 - < 0.5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Decreased thymus weight and microscopic and macroscopic changes in the thymus

Critical effects observed:

yes

Lowest effective dose / conc.:

0.4 mg/kg bw/day (nominal)

Organ:

other: thymus

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

not examined

Clinical signs:

not examined

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
The number of pups delivered per litter was comparable in all groups, 9.8, 10.8, 9.6 and 8.0 in the control, low-, mid- and high-dose groups respectively. The number of liveborn pups was 108, 129, 104 and 64 in the control, low-, mid- and high-dose groups and was considered to be statistically significantly decreased in the high dose group.
The number of stillborn pups in the control, low- and mid-dose groups were 0, in the high-dose group a statistically significant increase was noted, with 8 stillborns recorded (6 of these from the same litter, 1 each in two additional litters).
Pup mortality on post-natal day 4 (PN 4) was comparable in all groups except the high dose groups in which there was a statistically significant increase. 1, 2, 2 and 22 mortalities (incidences 0.9, 1.6, 1.9 and 34.0%) were recorded in the control, low-, mid- and high-dose groups respectively.
Only in the high-dose group, 3 litters were lost entirely between post-natal day 0 and 4.
The number of live pups per litter on PN 1 (9.8, 10.8, 9.6 and 8.0) and PN 4 (9.7, 10.6, 9.4 and 7.0), was not found to be statistically significantly different even though the number of live pups was decreased in the high-dose group on PN1 and 4.
No difference was observed in the sex ratio.

CLINICAL SIGNS (OFFSPRING)
On post-natal day 1, the number of runts was statistically significantly increased in the 250 mg/kg diet group. No other treatment related abnormalities were recorded.

BODY WEIGHT (OFFSPRING)
On post-natal day 1 and 4, a statistically significant decrease in pup bodyweight in the high-dose group was recorded (day 1 mean value for males+females in the high dose group was 4.54 g, compared to 5.08 in the control group). The pup weight change (PN 1-4) was significantly decreased in the male pups of the high-dose group (mean body weight gain of 1.38 g, compared to 2.56 g in the controls). No effects were noted in the other treatment groups.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic evaluation of the stillborn pups revealed 3 partially cannibalized pups and 3 autolytic pups in the high-dose group; the latter pups had no abnormalities. In addition 2 stillborn pup with no abnormalities in the high-dose groups were examined.

Reproductive effects observed:

not specified

Table 1: Test material concentration in experimental diets

Nominal concentration (mg/kg)	Mean Nominal Measured Concentration (mg/kg)	Percent of Nominal
0 (#1)	<0.05	NA
0 (#2)	<0.05	NA
0 (#3)	<0.05	NA
5 (#1)	4.52	90
5 (#2)	4.93	99
5 (#3)	4.40	88
25 (#1)	24.9	100
25 (#2)	26.5	106
25 (#3)	26.3	105
250 (#1)	247	99
250 (#2)	240	96
250 (#3)	244	98

#3: repeated analysis of batch no. 2

 

Table 2: Summary of relevant treatment related findings

Parameter	Dose levels
	5 mg/kg diet	25 mg/kg diet	250 mg/kg diet
Bodyweight: GD 21, PN 1 (females only)			Decreased
Bodyweight change: GD 14-21 (females only)			Decreased
Food consumption: PM 7-13 (males) GD 7-14, 14-21 and PN 1-4 (females only)			Decreased
Bilirubin (females only)			Increased
Alkaline phosphatase (males only)			Increased
Relative liver weight (females only)			Increased
Relative kidney weight (females only)			Increased
Absolute and/or relative thymus weight		Decreased	Decreased
Thymus: lymphoid depletion (males only)			Increased
Thymus: lymphoid depletion (females only)		Increased	Increased
Ovary: cysts (females only)			Increased
Liver: glycogenic vacuolation (females only)			Increased
Duration of gestation			Increased

 

Table 3: Reproduction and litter data (high dose group)

Parameter	High dose group	Control group
Mean duration of gestation (days)	21.7*	21
Number of females with liveborn pups	8	11
Gestation index (%)	80	100
* Statistically significantly different.

 

Conclusions:

Based on the effects noted in the thymus in both male and female rats in the 25 mg/kg diet groups, the NOAEL was concluded to be the lowest group tested, 5 mg/kg diet which was equivalent to 0.3-0.4 mg/kg bw/day for male animals and 0.3-0.5 mg/kg bw/day for female animals.
There are no relevant adverse findings concerning development and reproduction.

Executive summary:

The toxicological effects of the test substance and possible effects on reproduction was assessed in a repeated dose toxicity and reproductive and developmental screening study in rats. The study was performed in accordance with GLP and to the standardised guideline OECD 422.

Based on the effects noted in the thymus in both male and female rats in the 25 mg/kg diet groups, the NOAEL was concluded to be the lowest group tested, 5 mg/kg diet which was equivalent to 0.3-0.4 mg/kg bw/day for male animals and 0.3-0.5 mg/kg bw/day for female animals.

A NOAEL for reproductive toxicity was not considered necessary because the reproductive effects observed were non-specific and considered to be related to maternal toxicity.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

klimisch 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Justification for selection of Effect on fertility via oral route:
DOTAcAc hydrolyses in DOTO and 2,4-Pentadione

Effects on developmental toxicity

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Study period:

12 November 2003 to 8 March 2004

Data waiving:

other justification

Justification for data waiving:

other:

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10-11 weeks
- Weight at study initiation: The weight variation of the animals for each sex did not exceed 20 %.
- Diet: ad libitum
- Water: tap water ad libitum in polypropylene bottles

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 30-70 5
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

DOSE-RANGE FINDING TEST IN-LIFE DATES: From: 12 November 2003 To: 26 November 2003
MAIN TEST IN-LIFE DATES: From: 14 January 2004 To: 8 March 2004

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): The experimental diets were prepared once shortly before the study
- Mixing appropriate amounts with (Type of food): The test substance was weighed and placed in a small grinder. The tray was rinsed with food which was then also added to the grinder and mixed for 2 x 30 seconds. This mixture was moved to a Stephan cutter and the grinder was rinsed with food and moved to the cutter. Approximately 3 kg weighed food was mixed into the cutter for 2 x 2 minutes and moved. This was then moved to the Lödige cutter. The Stephan cutter was rinsed with approximately 3 kg of food and that was also moved to the Lödige cutter. Mixing was continued in the Lödige cutter for 2 minutes with the total amount of food.
- Storage temperature of food: <-18 °C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The feed was checked for homogenous distribution, stability and concentration of the test substance for all the doses in the dose-range finding study. The same dose preparation for the dose-range finding study was also used for the main test. The homogenous distribution and achieved concentration of the low dose in the main test was also analysed.
Directly after preparation of the diet for the dose-range finding study, samples for homogeneity and stability were taken. Five samples were taken (approximately 50 g each) to examine the homogeneity of the dose from the top centre, middle centre, bottom centre, left centre and right centre of the mixer. Secondly samples (around 50 g) were taken from the top centre part of the mixer to measure the stability. The samples taken for measurement of the homogeneity were also used for dose confirmation. In addition the content (achieved concentration) of the test substance in the batch of diet used in the main study. Diet samples were taken for analysis immediately after preparation and stored at – 18 °C.
Samples of the 0, 25, 75, 200 and 500 mg/kg diets from the dose-range finding study and doses of 0, 25 and 250 mg/kg from the main study as well as all related calibration samples were derivatised.
A calculated amount of internal standard solution (MHT, DHT and TTPT in methanol) was added to 2.0 g of diet in a 50 mL Corning tube. 10 mL of 100 % acetic acid was then added and the Corning tube was closed and shaken for 60 minutes (250 rpm). 10 mL of acetate buffer solution (pH 4.5) was added along with 10 mL methanol. 2.0 mL of 20 % (m/V) aqueous STEB solutions was added, followed by 10 mL hexane (containing naphthalene, approximately 0.1 mg/L). The tube was shaken for 15 minutes (250 rpm) then placed in an oven at 60 °C for 15 minutes. After phase separation, the hexane layer (approximately 3 mL) was removed and washed with 3 mL of 2 mol/L HCl (30 minutes shaking at 250 rpm). The hexane top layer was diluted with hexane: hexane extracts from sample with dose levels of 0, 25 and 75 mg/kg were diluted five times, the higher doses were diluted fifty times. The resulting solutions were transferred into an amber coloured glass vial and anaylysed using GC-MS.
The procedure for the samples from the main study at doses of 0 and 5 mg/kg followed the same derivatisation procedure, except the initial quantity of the diet was 5.0 g not 2.0 g
The concentration of organotin compounds in the extracts was determined using GC-MS. For calculation of the amount of DOTO in the samples, the peak area of DHT was used as an internal standard. Quantitiation was achieved using the calibration graphs constructed from the calibration solutions.
The following conditions were used:
- Column: Fused silica HP5 MS, 30 m, 0.25 mm ID, 0.25 µm film
- Precolumn: fused silica HP5 MS, 2.5 m, 0.25 mm ID, 0.25 µm film
- Column temperature: after 3 minutes at 45 °C at a rate of 5 °C/min to 80 °C; then at a rate of 15 °C/min to 260 °C; 15 min at 260 °C.
- Carrier: helium; 1.5 mL/min constant flow
- Injection volume: 1 µL
- Injection temperature: start at 60 °C, then at a rate of 14.5 °C/s to 300 °C; 5 min at 300 °C
- Injection method: splitless
- Ionisation: electron impact 70 eV
- Mass range: 60-600 amu
- Mass fragments used: DOT m/z = 375*; 263; 151, DHT 347*; 249; 179
Mass fragments marked with an asterisk were used for quantitation

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Until pregnancy occurred
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually

Duration of treatment / exposure:

Treated food was available ad libitum

Frequency of treatment:

Daily

Duration of test:

28 days

No. of animals per sex per dose:

22 males and 22 females in the 14-day dose range finding study (5 groups of 4 male and 4 female rats)
52 males and 52 females in the main study (4 groups of 12 male and 12 female rats)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Doses were selected on the results of the range finding test
- Rationale for animal assignment: Randomised

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Every morning throughout the study, and a second observation in the afternoon of working days.
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the first exposure and then once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of male and female rats were taken on day -2 (randomisation) and on days 0 (first day of dosing), 7 and 13 of the premating period.
Males were weighed weekly during the mating period until sacrifice. Females were weighed during mating (day 0, 7 and 13) and mated females were weighed on day 0, 7, 14 and 21 during presumed gestation and on day 1 and 4 of lactation. All animals were weighed at sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, also calculated as g/animal/day

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the premating period
- Anaesthetic used for blood collection: Yes, CO2/02 anaesthesia
- Anit-coagulant: K2-EDTA
- Animals fasted: Yes, overnight
- How many animals: 5 rats/group
- Parameters checked:
Haemoglobin
Packed cell volume
red blood cell count
reticulocytes
Total white blood cell count
Prothrombin time
Thrombocyte count
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the premating period
- Anaesthetic used for blood collection: Yes, CO2/02 anaesthesia
- Animals fasted: Yes, overnight
- How many animals: 5 rats /group
- Parameters checked:
Fasting glucose
Alkaline phosphatase activity (ALP)
Aspartate aminotransferace activity (ASAT)
Alanine aminotransferace activity (ALAT)
Gamma glutamyl transferase activity (GGT)
Total protein
Albumin
Ratio albumin to globulin
Urea
Creatinine
Bilirubin (total)
Cholesterol (total)
Triglycerides
Phospholipids
Calcium (Ca)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Inorganic phosphate

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Arena testing was performed prior to the first exposure and then once weekly until the end of dosing and in females until the end of lactation. Males and females selected for the functional observation battery test (FOB) and spontaneous activity measurements were excluded from the final arena testing.
At the end of the study, FOB test and spontaneous motor activity measurements were performed on day 27 for males and on post natal day for for females.
- Dose groups that were examined: All animals were subject to the arena testing. For the other two tests, 5 animals were randomly selected from each dose group.
- Battery of functions tested:
Autonomic: lacrimation, salivation, pupil response to light, palpebral closure, piloerection, defecation and urination
Neuromuscular: gait, mobility, forelimb and hindlimb gripstrength, landing foot splay, righting reflex
Sensorimotor: response to tail pinch, click, tough and approach of a visual object
Convulsive: clonic and tonic movements
Excitability: ease of removal, handling reactivity, arousal and vocalisations
Activity: rearing and motor activity
Physiological: body temperature

SACRIFICE
- Maternal animals: Sperm positive females that were not pregnant were killed 25 days after copulation, mothers with litters were killed on post natal day 4.

GROSS PATHOLOGY: Yes
The following were taken from all animals:
Ovaries
Uterus
Organs and tissues showing macroscopic abnormalities
The following were taken from 5 rats/group:
Adrenals
Axillary lymph node
Bone marrow (femur)
Brain
Caecum
Coagulation glands
Colon
Duodenum
Eyes
Heart
Jejunum
Lungs
Kidneys
Liver
Mammary gland (females only)
Mesentric lymph node
Parathyroids
Peyer's patches
Pituitary
Rectum
Sciatic nerve
Spinal cord
Spleen
Stomach
Thymus
Thyroids
Trachea
Urinary bladder

The following organs were weighed:
Adrenals
Brain
Heart
Kidneys
Liver
Spleen
Thymus

HISTOPATHOLOGY: Yes Microscopic examination was performed on the collected organs of all rats in the control and high-dose group.
The liver and ovaria of females and the thymus of the male and female rats in the low and mid-dose groups were also evaluated
The following tissues, though collected were not subject to histopathological examination:
Coagulation glands
Mammary gland (females only)
In addition, reproductive organs of females that were non-mated or non-pregnant of the mid and low dose groups were microscopically examined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

- External examinations: Yes: all still born pups and pups that died during lactation

Statistics:

Clinical findings and histopathological changes of the maternal animals were evaluated using Fisher’s exact probability test.
Number of implantation sites, live and dead pups were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann Whitney U-test.
Bodyweight, bodyweight gain, organ weights, food consumption, red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values and organ weights were assessed by one-way ANOVA followed by Dunnett’s multiple comparison tests.
Reticulocytes and relative differential white blood cell counts were assessed using Kruskal-Wallis non-parametric ANOVA followed by Mann-Whitney U-tests.
The results of the functional observations were measured on different scales. Continuous measurements were analysed by one-way analysis of variance at each time point, if found to be statistically significant, a post-hoc group comparison was performed. Rank order data were analysed by Kruskal-Wallis analysis of variance at each test time point, followed by planned multiple comparisons between dose groups were a significant results occurred. Categorical data were assessed using Pearson chi-square analysis.
Motor activity data were assessed by one-way analysis of variance at each time point with a post-hoc group comparison performed on significant results.
All tests were two sided and the level of probability p<0.05 was considered as significant. Effects of treatment on habituation were analysed by repeated measures of analysis variance in five 6 minute time blocks. Statistical evaluations on pup variables were considered on a litter basis. Additional evaluations on a pup basis were performed to identify any specific dose-related effect that may have occurred.

Indices:

Sex ratio day n = (number of live male pups on day n/number of live pups on day n) x 100

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
One female in the high dose group was found dead on gestation day 24; this animal was pregnant and 11 dead foetuses were found in the uterus.
The only finding during the gestation period (gestation day 21) was a sparsely haired animal in the 250 mg/kg group. During the lactation period, sparsely haired animals were noted in the control (n = 1), 5 mg/kg (n = 1) and in the 250 mg/kg group (n = 1). No other findings were noted.

BODY WEIGHT AND WEIGHT GAIN
Mean bodyweights of the dams of the high-dose group was statistically significantly decreased on gestation day 21 (8.5% lower than controls) and post-natal day 1 (9.3% lower than controls). Mean bodyweight changes of the dams of the high-dose group were more markedly affected during pregnancy, as the overall mean weight gain for the control animals accounted to 74.31 g, whereas that in the high-dose animals it was only 51.43 g (approx. 31% lower); during gestation day 14 to 21, the reduction in mean weight gain in the high-dose animals attained statistical significance, compared to controls, and was approximately 52% lower. Bodyweights and bodyweight change of the dams of all the treated groups were comparable to the controls group at all other times of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
During the gestation period and lactation period (gestation days 7-14 and 14-21 and post-natal days 1-4), food consumption (expressed as g/animal/day and g/kg bodyweight/day) of the dams in the high-dose group was significantly decreased. Considering the entire pregnancy period, high-dose animals ate approx. 13% less food than controls (in terms of g/kg/day), and approx. 24% less food than controls during the 4-day lactation period. No other treatment-related effects were observed.

- Females (listed as low, mid and high dose groups; 5, 25 and 250 mg/kg diet respectively)
premating days 0-7: 0.4, 1.7 and 17.4 mg/kg bodyweight/day
premating days 7-13: 0.3, 1.6 and 15.4 mg/kg bodyweight/day
gestation days 0-7: 0.4, 2.0 and 17.4 mg/kg bodyweight/day
gestation days 7-14: 0.4, 1.9 and 16.7 mg/kg bodyweight/day
gestation days 14-21: 0.3, 1.4 and 11.2 mg/kg bodyweight/day
Post-natal days 1-4: 0.5, 2.4 and 17.4 mg/kg bodyweight/day

HAEMATOLOGY
All treated groups were found to be comparable to controls

CLINICAL CHEMISTRY
Bilirubin (µmol/L) was found to be statistically significantly increased in the high-dose females. this finding were considered to be treatment related. Other effects such as the statistically significant increase in calcium in the 5 mg/kg group was not considered to be related to treatment. No other changes were observed.

NEUROBEHAVIOUR
No treatment-related effects were observed.

ORGAN WEIGHTS
The absolute and relative weight of the female animals of the high dose group was significantly decreased (-69 and -66%, respectively, compared to controls). In the mid-dose group the relative thymus weight was also statistically significantly decreased (-36% compared to controls).
In the female animals of the high-dose group, the relative kidney and liver weights were statistically significantly increased (+14 and +22%, respectively, compared to controls).
No other effects were observed.

GROSS PATHOLOGY
At necropsy, a decrease in thymic size was seen in all animals in the 250 mg/kg diet groups, 11 animals in the 25 mg/kg diet group, 7 animals in the 5 mg/kg diet group and 5 animals in the control group.
Examination of the female that was found dead revealed hydrothorax, haemorrhagic lungs, dilation of the vena cava and haemorrhagic discharge in the vagina, these were considered to be indicative of problems during parturition.

HISTOPATHOLOGY
Microscopic evaluation of the thymus revealed moderate to very severe lymphoid depletion in all animals of the 250 and 25 mg/kg diet groups. Lymphoid depletion was characterised by a decrease in the thymic lobules due to an extensive loss of cortical and medullary small lymphocytes. The distinction between the cortical and medullary areas was unclear. In the more extreme effects observed, the cortex was very small, or absent. The remaining lymphoid cells visible in the cortical areas were mainly lymphoblasts. Lymphoblastic cells and reticuloepithelial cells had increased, and/or higher numbers of these cells were visible due to the disappearance of small lymphocytes and the collapse of the thymic stroma. In 3 high-dose animals, lymphoid depletion was accompanied by lymphoid depletion in the PALS (periateriolar lymphocyte sheath areas) in the spleen. The macroscopically observed thymi in 5 control and 7 low dose females exhibited no microscopic abnormalities. In the thymi of the 2 control and 2 low dose females pregnancy/lactation involution was observed. The thymic lobules were decreased in size but exhibited normal structure with the histological appearance of age-involution. Increased glycomeric vacuolation, viz moderate versus very slight was seen in the liver of 4 high dose females and was considered to be a potential cause of the increased weight.
Examination of the reproductive organs revealed a statistically significant increased in the incidence of cysts in the ovaries of 8 high-dose females.

Dose descriptor:

NOAEL

Effect level:

> 0.3 - < 0.5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
VIABILITY
The number of females with liveborn pups was 11, 11, 11 and 8 for the control, low, mid and high dose respectively.
One female was found dead in the high dose group on gestation day 24; the animal was pregnant and 11 dead foetuses were discovered in the uterus. Stillborn pups were observed in the high dose in three litters. In the high-dose group 1 female with all stillborn pups was observed.
Pre-implantation loss was 8.4. 2.2. 12.3 and 6.2% for the control, low-, mid- and high-dose groups and was considered comparable.
Pos-implantation loss was 9.8, 11.6, 7.5 and 38.7% for the control, low-, mid- and high-dose groups respectively. The post-implantation loss recorded in the high-dose group was considered to be statistically significantly increased.
The number of pups delivered per litter was comparable in all groups, 9.8, 10.8, 9.6 and 8.0 in the control, low-, mid- and high-dose groups respectively. The number of liveborn pups was 108, 129, 104 and 64 in the control, low-, mid- and high-dose groups and was considered to be statistically significantly decreased in the high dose group.
The number of stillborn pups in the control, low- and mid-dose groups were 0, in the high-dose group a statistically significant increase was noted, with 8 stillborns recorded (6 of these from the same litter, 1 each in two additional litters).
Pup mortality on post-natal day 4 (PN 4) was comparable in all groups except the high dose groups in which there was a statistically significant increase. 1, 2, 2 and 22 mortalities (incidences 0.9, 1.6, 1.9 and 34.0%) were recorded in the control, low-, mid- and high-dose groups respectively.
Only in the high-dose group, 3 litters were lost entirely between post-natal day 0 and 4.
The number of live pups per litter on PN 1 (9.8, 10.8, 9.6 and 8.0) and PN 4 (9.7, 10.6, 9.4 and 7.0), was not found to be statistically significantly different even though the number of live pups was decreased in the high-dose group and PN1 and 4.
No difference was observed in the sex ratio

CLINICAL SIGNS (OFFSPRING)
On post-natal day 1, the number of runts was statistically significantly increased in the 250 mg/kg diet group. No other treatment related abnormalities were recorded.

BODY WEIGHT (OFFSPRING)
On post-natal day 1 and 4, a statistically significant decrease in pup bodyweight in the high-dose group was recorded (day 1 mean value for males+females in the high dose group was 4.54 g, compared to 5.08 in the control group). The pup weight change (PN 1-4) was significantly decreased in the male pups of the high-dose group (mean body weight gain of 1.38 g, compared to 2.56 g in the controls). No effects were noted in the other treatment groups.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic evaluation of the stillborn pups revealed 3 partially cannibalized pups and 3 autolytic pups in the high-dose group; the latter pups had no abnormalities. In addition 2 stillborn pups with no abnormalities in the high-dose groups were examined.

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Test material concentration in experimental diets

Nominal concentration (mg/kg)	Mean Nominal Measured Concentration (mg/kg)	Percent of Nominal
0 (#1)	<0.05	NA
0 (#2)	<0.05	NA
0 (#3)	<0.05	NA
5 (#1)	4.52	90
5 (#2)	4.93	99
5 (#3)	4.4	88
25 (#1)	24.9	100
25 (#2)	26.5	106
25 (#3)	26.3	105
250 (#1)	247	99
250 (#2)	240	96
250 (#3)	244	98
#3: repeated analysis of batch no. 2

 

Table 2: Summary of relevant treatment related findings

Parameter	Dose levels
	5 mg/kg diet	25 mg/kg diet	250 mg/kg diet
Bodyweight: GD 21, PN 1 (females only)			Decreased
Bodyweight change: GD 14-21 (females only)			Decreased
Food consumption: PM 7-13 (males) GD 7-14, 14-21 and PN 1-4 (females only)			Decreased
Bilirubin (females only)			Increased
Alkaline phosphatase (males only)			Increased
Relative liver weight (females only)			Increased
Relative kidney weight (females only)			Increased
Absolute and/or relative thymus weight		Decreased	Decreased
Thymus: lymphoid depletion (males only)			Increased
Thymus: lymphoid depletion (females only)		Increased	Increased
Ovary: cysts (females only)			Increased
Liver: glycogenic vacuolation (females only)			Increased
Post-implantation loss			Increased
Number of stillborn pups			Increased
Pup mortality PN 4			Increased
Pup weight PN 1			Decreased
Number of runts PN 1			Increased

 

Table 3: Litter data (high dose group)

Parameter	High dose group	Control group
Number of females with liveborn pups	8	11
Post-implantation loss (%)	38.7*	9.8
Mean number of pups delivered	8.0	9.8
Mean number of live pups/litter PN1	8.0	9.8
Mean number of live pups/litter PN 4	7.0	9.7
Total number of stillborn pups	8*	0
Pup mortality PN 4 (%)	34*	0.9
Pup weight PN 1 (g)	4.5*	5.1
Pup weight PN 4 (g)	6.5	7.6
Percentage of runts PN 1 (%)	34*	2
* Statistically significantly different.

Conclusions:

Based on the effects noted in the thymus in both male and female rats in the 25 mg/kg diet groups, the NOAEL was concluded to be the lowest group tested, 5 mg/kg diet which was equivalent to 0.3-0.4 mg/kg bw/day for male animals and 0.3-0.5 mg/kg bw/day for female animals.
There are no relevant adverse findings concerning development and reproduction.

Executive summary:

The developmental effects of the test substance was assessed in a repeated dose toxicity and reproductive and developmental screening study in rats. The study was performed in accordance with GLP and to the standardised guideline OECD 422. Only one death was noted during the study.

Based on the effects noted in the thymus in the 25 mg/kg diet groups, the NOAEL for general toxicity was concluded to be the lowest group tested, 5 mg/kg diet which was equivalent to 0.3-0.5 mg/kg bw/day.

A NOAEL for developmental toxicity was not considered necessary because the reproductive effects observed were non-specific and considered to be related to metarnal toxicity.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conduction and documentation of study very acceptable

Justification for type of information:

Tin, dioctylbis(2,4-pentanedionato-κO2,κO4)- hydrolyses with humidity into Dioctyltinoxide and 2,4-Pentadione. There is an evidence that the hydrolysis product 2,4-Pentadion cause an adverse effect in worker (US EPA)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

Timed-pregnant Fischer F-344 rats (Harlan Fischer F-344/HarBR) were exposed to 2,4-pentanedione vapour by inhalation on gestational days (gd) 6 to 15 at exposure target concentrations of 0, 50, 200 and 400 ppm (0, 52.7, 202 and 398 ppm mean analytical concentrations, respectively) to evaluate the embryotoxic and fetotoxic (including tera-togenic) potential of the TS administered during organogenesis. The day a copulation plug was found was designated gestational day (gd) 0. Twenty-five plug-positive fe-males were assigned to each experimental group. Clinical observations were recorded daily, and maternal body weights were taken on gd 0, 6, 9, 12, 15 and 18. At scheduled necropsy on gd 21 (CO2 asphyxiation), dams were evaluated for body weight, liver and thymus weights, gravid uterine weight, and status of implantation sites (i.e. resorptions, dead fetuses, live fetuses). Maternal brains were removed, fixed and examined histopa-thologically. Live fetuses were dissected from the uterus, counted, weighed and sexed and examined for external abnormalities. weighed and sexed and examined for external abnormalities. Approximately one-half of the live fetuses in each litter was examined for visceral abnormalities. These fetuses were then decapitated and their heads fixed in Bouins solution and examined for soft tissue craniofacial malformations. The remaining intact fetuses in each litter were eviscerated, fixed in alcohol, stained with alizarin red S, and examined for skeletal defects and deficits.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation:
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- Verification of same strain and source of both sexes: [yes / no (explain)]
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy
- Any other deviations from standard protocol:

Duration of treatment / exposure:

gestational days (GD) 6-15

Frequency of treatment:

6 h/day consecutive days

Duration of test:

13 days (treatment), animals were sacrificed on GD 21

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Dose descriptor:

NOAEC

Effect level:

200 ppm

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEC

Effect level:

400 ppm

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: educed fetal weights; reduced fetal ossification in the 200 and 400 ppm group

Dose descriptor:

NOAEC

Effect level:

50 ppm

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Dose descriptor:

LOAEC

Effect level:

200 ppm

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Dose descriptor:

NOAEC

Effect level:

400 ppm

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEC

Effect level:

400 ppm

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

.

Conclusions:

Based on a significantly reduced body weight gain in the 400 ppm exposure group the NOAEC/LOAEC derived for maternal toxicity is 200 and 400 ppm, respectively. The NOAEC/LOAEC for developmental toxicity is 50 and 200 ppm, respectively, which is based on reduced fetal weights in male fetuses at 200 ppm and in male and female fe-tuses at 400 ppm and a consistent pattern of reduced fetal ossification at 400 ppm. The NOAEL for embryotoxicity and teratogenicity is 400 ppm (highest dose tested).

Executive summary:

Groups of 25 timed-pregnant Fischer F-344 rats (Harlan Fischer F-344/HarBR) were exposed to 2,4-pentanedione vapour by inhalation on gestational days (gd) 6 to 15 at exposure target concentrations of 0, 50, 200 and 400 ppm (0, 52.7, 202 and 398 ppm mean analytical concentrations, respectively) to evaluate the embryotoxic and fetotoxic (including teratogenic) potential of the test substance administered during organogenesis. The day a copulation plug was found was designated gestational day (gd) 0. Twenty-five plug-positive females were assigned to each experimental group. Clinical observations were recorded daily, and maternal body weights were taken on gd 0, 6, 9, 12, 15 and 18. At scheduled necropsy on gd 21 (CO2 asphyxiation), dams were evaluated for body weight, liver and thymus weights, gravid uterine weight, and status of implantation sites (i.e. resorptions, dead fetuses, live fetuses). Maternal brains were removed, fixed and examined histopathologically. Live fetuses were dissected from the uterus, counted, weighed and sexed and examined for external abnormalities. weighed and sexed and examined for external abnormalities. Approximately one-half of the live fetuses in each litter was examined for visceral abnormalities. These fetuses were then decapitated and their heads fixed in Bouins solution and examined for soft tissue craniofacial malformations. The remaining intact fetuses in each litter were eviscerated, fixed in alcohol, stained with alizarin red S, and examined for skeletal defects and deficits.

There was no maternal mortality in this study. Significantly reduced body weight gain at 400 ppm; liver weight significantly increased at 200 ppm but no further significant effects were determined. Maternal toxicity was indicated by reduced body weights on gd 9, 12, 15, and 18 but not on gd 21, and reduced weight gain for the intervals gd 6-9, 6-12, 6-15 (exposure period) and gd 6-18, but not for the post-exposure period (gd 15-21). There were no treatment-related effects on maternal liver, thymus or gravid uterine weight, or on body weight (absolute or corrected for gravid uterus) at sacrifice; histologic examination of the maternal brains showed no pathological effects related to treatment. For fetal toxicity significant reduction in female body weight per litter at 400 ppm (all fetuses, males and females approximately 10 %) and at 200 ppm (all fetuses, and males but not females approximately 3 %), one visceral variation (partial fetal atelectasis) significantly increased at 400 ppm were observed. 17 out of 79 observed skeletal variation exhibited significant changes in incidence and indicated a consistent pattern of reduced ossification in the 400 ppm group (for example poorly or unossified phalanges, unossified cervical or poorly ossified thoracic centrum). No differences were observed among the groups in the incidence of external, visceral or skeletal malformations; no further treatment related effects. There were also no effects of treatment on the number of ovarian corpora lutea, of total, non-viable or viable implantations per litter, or on pre- or post-implantation loss or on sex ratio. There were no maternal deaths, early deliveries or abortions. Pregnancy rate was high and equivalent across all treatment groups. One dam each at 0, 50 and 200 ppm carried a totally resorbed litter on gd 21. Two dams at 400 ppm had totally resorbed litters on gd 21. Clinical observations were made daily throughout the study. Most of the observations were limited to the eyes, nose and blood at the vaginal orifice and only in a few dams per group. In addition, urogenital area wetness was present in a few dams only at 0, 50, 200 ppm (not at 400 ppm). At sacrifice on gd 21, there was no effect of exposure on maternal body weight, maternal body weight corrected for gravid uterine weight or on absolute or relative (to corrected body weight) thymus weight. Absolute and relative liver weight was elevated at 200 but not at 400 ppm. Administration of 2,4-pentanedione vapour by inhalation to timed-pregnant Fischer F-344 rats during organogenesis at 0, 50, 200 and 400 ppm resulted in maternal toxicity at 400 ppm. Fetotoxicity was observed at 200 and 400 ppm in terms of reduced fetal weights per litter (approximately 3 and 10 %, respectively) and at 400 ppm in terms of a consistent pattern of reduced fetal ossification. There was no evidence of embryotoxicity or teratogenicity at any exposure concentrations employed, including those which produced maternal toxicity. Based on a significantly reduced body weight gain in the 400 ppm exposure group the NOAEC/LOAEC derived for maternal toxicity is 200 and 400 ppm, respectively. The NOAEC/LOAEC for developmental toxicity is 50 and 200 ppm, respectively, which is based on reduced fetal weights in male fetuses at 200 ppm and in male and female fetuses at 400 ppm and a consistent pattern of reduced fetal ossification at 400 ppm. The NOAEC for embryotoxicity and teratogenicity is 400 ppm (highest dose tested). 

Reference 3

Endpoint:

developmental toxicity

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION OF READ ACROSS
Hydrolysis under stomach condition (pH 1.2) of Diotyltin bis(pentadione) into Dioctyltin oxide and pentadione (cp. Section basic toxicokinetics:)

Study for justification of read across is link via cross reference.

Reason / purpose for cross-reference:

other: Hydrolysis under stomach condition (pH 1.2)

Reason / purpose for cross-reference:

other: immunotoxicologial part of study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not applicable

Remarks:

additional immunotixicity, tin in feces and plasma, zin in plasma

Principles of method if other than guideline:

additional immunotixicity, tin in feces and plasma, zin in plasma

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Route of administration:

oral: feed

Vehicle:

other: test substance was applicated in diet

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

yes, see attached report

Details on mating procedure:

not relevant for OECD 414

Dose / conc.:

0 mg/kg diet

Remarks:

0 mg/kg bw / say

Dose / conc.:

5 mg/kg diet

Remarks:

0.4 mg/kg bw / say

Dose / conc.:

25 mg/kg diet

Remarks:

1.8 mg/kg bw / say

Dose / conc.:

200 mg/kg diet

Remarks:

11.8 mg/kg bw / say

Control animals:

yes, plain diet

Maternal examinations:

Dioctyltin Oxide-related changes in the thymus weights were recorded in toxicity females, compared with concurrent controls.
Group mean thymus:body weight ratios and unadjusted thymus weights were lower for toxicity females administered 200 mg/kg diet, compared with concurrent controls

Ovaries and uterine content:

findings in other tissues [beyond thymus] were generally infrequent, of a minor nature and consistent with the usual pattern of findings in rats of this strain and age

Fetal examinations:

Minor increases in some variations were recorded in fetuses/litters from high dose group; these included sternebrae with misaligned ossification centres, incomplete ossification of cervical centrum (p<0.05) and split in xyphoid cartilage, with the latter also showing an increase in intermediate dose fetuses/litters. A dose-relationship was not apparent and these minor changes were considered to be of no toxicological significance.

Any other intergroup differences, including those achieving statistical significance, were considered to be due to normal biological variation

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Description (incidence and severity):

no data reported in raw draft data

Clinical biochemistry findings:

not examined

Description (incidence and severity):

no data reported in raw draft data

Urinalysis findings:

not examined

Description (incidence and severity):

no data reported in raw draft data

Behaviour (functional findings):

no effects observed

Immunological findings:

effects observed, treatment-related

Description (incidence and severity):

The results indicated that test article-related changes were limited to a slight to marked, transient, increase in circulating CD25 positive B and T lymphocytes noted at the 11.8 mg/kg/day dose level only. Results at necropsy indicate that this finding was reversible.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Dioctyltin Oxide-related changes in the thymus weights were recorded in toxicity females, compared with concurrent controls.
Group mean thymus:body weight ratios and unadjusted thymus weights were lower for toxicity females administered 200 mg/kg diet, compared with concurrent controls

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Upon macroscopic examination, Dioctyltin Oxide-related findings were noted in the thymus.
Small thymus was recorded in some toxicity females administered 200 mg/kg diet, which generally correlated with microscopic findings.
No other macroscopic findings considered related to Dioctyltin Oxide were recorded.
Other tissues were macroscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Upon microscopic examination, Dioctyltin Oxide-related findings were recorded in the thymus of toxicity animals.
An increased incidence and severity of thymus atrophy was recorded in most females administered 200 mg/kg diet

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

no data reported in raw draft data

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): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): not relevant in an OECD 414 study

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

see attached summary and raw fata fetel
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): see attached summary and raw fata fetel

Reduction in number of live offspring:

not examined

Description (incidence and severity):

see attached summary and raw fata fetel

Changes in sex ratio:

no effects observed

Description (incidence and severity):

see attached summary and raw fata fetel

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

see attached summary and raw fata fetel

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

see attached summary and raw fata fetel

External malformations:

no effects observed

Description (incidence and severity):

see attached summary and raw fata fetel

Skeletal malformations:

no effects observed

Description (incidence and severity):

see attached summary and raw fata fetel

Visceral malformations:

no effects observed

Description (incidence and severity):

see attached summary and raw fata fetel

Other effects:

not examined

Description (incidence and severity):

no data reported in raw draft data

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 11.8 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

The LOAEL for acute immune toxicity for Dioctyltin oxide was determined to be 11.8 mg / kg bw7day thus a NOAEL of 1.8 mg/kg bw7day results foaternnal toxicity, with decrease of thymus. No adverse effects relating to reproduction/development

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Additional information

1. Reproduction and development toxicity

According to the ICH Guideline S5(R2) Part 1 [XX] the aim of reproduction studies is the reveal any effect on mammalian reproduction.

The results should be related to all other available data to determine whether potential reproductive risks pharmacological and toxicological to humans are greater, lesser or equal to those posed by other toxicological manifestations.

Toxicological manifestation in item of drugs are defined as intended or adverse effect, in contrast for chemical safety every toxicological manifestation is to assess as an adverse effect (compare section 4.1.2 – maternal toxicity).

The term reproduction covers the following stages:

• Premating to conception (adult male and female reproductive functions, development and maturation of gametes, mating behavior, fertilisation).

• Conception to implantation (adult female reproductive functions, pre-implantation development, implantation).

• Implantation to closure of the hard palate (adult female reproductive functions, embryonic development, major organ formation).

• Closure of the hard palate to the end of pregnancy (adult female reproductive functions, fetal development and growth, organ development and growth).

• Birth to weaning (adult female reproductive functions, neonate adaption to extrauterine life, pre-weaning development and growth).

• Weaning to sexual maturity (post-weaning development and growth, adaption to independent life, attainment of full sexual function).

F

2. Maternal toxicity

Primarily, reproductive toxicity follows the same rules as other types of toxicity and is subject to the same general principles as laid down by Paracelsus in the 16^thCentury “All substances are poisons; there is none which is not a poison. The right dose differentiates a poison from a remedy.’ In addition however, some substances have the potential to selectively affect the developing embryo to cause malformations at dose levels below those causing any other adverse effects.

This indicates the Directive 69/548/EEC [XX] with:

“Classification of chemicals as toxic to reproduction is intended to be used for chemicals which have an intrinsic or specific property to produce such toxic effects. Chemicals should not be classified as toxic to reproduction where such effects are solely produced as a non-specific secondary consequence of other toxic effects. Chemicals of most concern are those which are toxic to reproduction at exposure levels which do not produce other signs of toxicity.”

The UNECE discussed in their Final Draft for GHS WEB for Reproductive Toxicity [XX] maternal toxicity and criteria therefor:

16. Development of the offspring throughout gestation and during the earlypostnatal stages can be influenced by toxic effects in the mother either through non-specific mechanisms related to stress and the disruption of maternal homeostasis, or by specific maternally mediated mechanisms. So, in the interpretation of the developmental outcome to decide classification for developmental effects it is important to consider the possible influence of maternal toxicity. This is a complex issue because of uncertainties surrounding the relationship between maternal toxicity and developmental outcome. Expert judgment and a weight of evidence approach, using all available studies, should be used to determine the degree of influence that should be attributed to maternal toxicity when interpreting the criteria for classification for developmental effects. The adverse effects in the embryo/fetus should be first considered, and then maternal toxicity, along with any other factors which are likely to have influenced these effects, as weight of evidence, to help reach a conclusion about classification.

17. Based on pragmatic observation, it is believed, that maternal toxicity may, depending on severity, influence development via non-specific secondary mechanisms, producing effects such as depressed fetal weight, retarded ossification, and possibly resumptions and certain malformations in some strains of certain species. However, the limited numbers of studies which have investigated the relationship between developmental effects and general maternal toxicity have failed to demonstrate a consistent, reproducible relationship across species. Developmental effects which occur even in the presence of maternal toxicity are considered to be evidence of developmental toxicity, unless it can be unequivocally demonstrated on a case by case basis that the developmental effects are secondary to maternal toxicity. Moreover, classification should be considered where there is significant toxic effect in the offspring, e.g. irreversible effects such as structural malformations, embryo/fetal lethality, significant post-natal functional deficiencies.

18. Classification should not automatically be discounted for chemicals that produce developmental toxicity only in association with maternal toxicity, even if a specific maternally mediated mechanism has been demonstrated. In such a case, classification in Category 2 may be considered more appropriate than Category 1. However, when a chemical is so toxic that maternal death or severe inanition results, or the dams are prostrate and incapable of nursing the pups, it may be reasonable to assume that developmental toxicity is produced solely as a secondary consequence of maternal toxicity and discount the developmental effects. Classification may not necessarily be the outcome in the case of minor developmental changes e.g. small reduction in fetal/pup body weight, retardation of ossification when seen in association with maternal toxicity.

19. Some of the end points used to assess maternal toxicity are provided below. Data on these end points, if available, needs to be evaluated in light of their statistical or biological significance and dose response relationship.

Maternal Mortality: An increased incidence of mortality among the treated dams over the controls should be considered evidence of maternal toxicity if the increase occurs in a dose related manner and can be attributed to the systemic toxicity of the test material. Maternal mortality greater than 10% is considered excessive and the data for that dose level should not normally be considered for further evaluation.

Mating Index(no. animals with seminal plugs or sperm/no. mated x 100)1

Fertility Index(no. animals with implants/no. of matings x 100)1

Gestation Length(if allowed to deliver)

Body Weight and Body Weight Change: Consideration of the maternal body weight change and/or adjusted (corrected) maternal body weight should be included in the evaluation of maternal toxicity whenever such data are available. The calculation ofan adjusted (corrected) mean maternal body weight change, which is the difference between the initial and terminal body weight minus the gravid uterine weight (or alternatively, the sum of the weights of the fetuses), may indicate whether the effect is maternal or intrauterine. In rabbits, the body weight gain may not be useful indicators of maternal toxicity because of normal fluctuations in body weight during pregnancy. Food and Water Consumption (if relevant). The observation of a significant decrease in the average food or water consumption in treated dams compared to the control group may be useful in evaluating maternal toxicity, particularly when the test material is administered in the diet or drinking water. Changes in food or water consumption should be evaluated in conjunction with maternal body weights when determining if the effects noted are reflective of maternal toxicity or more simply, unpalatability of the test material in feed or water.

Clinical evaluations:(including clinical signs, markers, hematology and clinical chemistry studies). The observation of increased incidence of significant clinical signs of toxicity in treated dams relative to the control group may be useful in evaluating maternal toxicity. If this is to be used as the basis for the assessment of maternal toxicity, the types, incidence, degree and duration of clinical signs should be reported in the study. Examples of frank clinical signs of maternal intoxication include: coma, prostration, hyperactivity, loss of righting reflex, ataxia, or labored breathing.

Post-mortem data:Increasedincidence and/or severity of post-mortem findings may be indicative of maternal toxicity. This can include gross or microscopic pathological findings or organ weight data, e.g., absolute organ weight, organ-to-body weight ratio, or organ-to-brain weight ratio. When supported by findings of adverse histopathological effects in the affected organ(s), the observation of a significant change in the average weight of suspected target organ(s) of treated dams, compared to those in the control group, may be considered evidence of maternal toxicity.

In the majority of guidelines only serve maternal effects would be registered, with exception of the OECD 422 Guideline.

Thus acute maternal responses may remain undetected in numerous cases [Influence of Maternal Toxicity in Studies on Developmental Toxicity 2 March 2004, Berlin - Workshop Report No. 4]

The following table lists relevant guidelines for development and reproduction toxcitiy and the “endpoint” acute maternal toxcity.

Table X - uidelines for development and reproduction toxcitiy and the “endpoint” acute maternal toxcity

Guideline No.	Description Guideline	Evaluation of maternal toxicity
OECD 414	Prenatal Developmental Toxicity Study	- Body weight - Food consumption - Clinical observation
OECD 415	One-Generation Reproduction Toxicity Study	- Body weight - Food consumption - Clinical observation -necropsy findings - organe weights - microscopic findings
OECD 416	Two-Generation Reproduction Toxicity Study	- Body weight - Food consumption - Clinical observation -necropsy findings - organe weights - microscopic findings
OECD 421	Reproduction/Developmental Toxicity Screening Test	- Body weight - Food consumption - Clinical observation -necropsy findings - organe weights - microscopic findings
OECD 422	Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test	- Body weight - Food consumption - Clinical observation -necropsy findings - organe weights - microscopic findings -haematological tests -clinical biochemistry tests

U.S. EPA suppose that it is expedient to add parameters like enzyme markers, haematology and clinical chemistry to all development and reproduction toxcitiy studies [EPA. 1991. Guidelines for developmental toxicity risk assessment. Federal Register 56(234): 63798-63826].

The end points “Enzyme markers” and “Clinical chemistries” are not a mandatory part of the data to be provided under the revised test guidelines (e.g., OECD 414; 2001). However, the value of such information has been recognized, and appropriate measurements, amongst others, are recommended in the ECETOC monograph (2002). Such studies have been performed on a case-bycase basis. A very recent Technical Guidance Document on Risk Assessment (European Commission, 2003) advocates “ideally, toxicokinetic and metabolism data should form part of the basis for a decision on the route of exposure used in the tests for reproductive toxicity.”[Influence of Maternal Toxicity in Studies on Developmental Toxicity 2 March 2004, Berlin - Workshop Report No. 4]

In general such information is not determined for industrial chemicals and commonly does not exist.

4. Primary and secondary developmental toxicity

   The terminology “direct” or “primary” developmental toxicity describes the circumstances in which a toxic agent or a metabolite of the agent acts directly on the conceptus to produce an adverse effect.

“Indirect” or “secundary” developmental toxiciy describes the circumstances in which the toxicant affects somse aspects of maternal function (compare section 4.1.3) that in turn adversly affects development. In literature some this the terminology “maternally mediate developmental toxicity is used [Daston, Developmental toxicology 2 (1994): 189-212.].

The mammalian embryo ist fully dependent on its mother to provide a healthy environment in which to develop. This involves an adequate supply of oxygen and nutrients to support intermediary metabolism and growth; adequate removal of carbon dioxide and metabolic wastes to maintain pH and prevent accumulation of toxic levels of metabolic products; an appropriate body temperature. Postnatally, the mother continues to provide the nutrutional needs of ther offspring via lactation. In rodent species, the mother also controls the body temperature of ther offspring until thy become homeothermic, and must stimulate micturition and defection for the first two weeks after birh. Other behavioral aspects of maternal caremay be subtle and difficult to measure, but clearly important for the infant´s well-being. Therefore, perturbation of uterine blood flow, maternal cadiocascular, pulmonary and renal function, nutritional state, placenta interfrity, endocrine factors that maintain pregancy, thermoregulation, maternal behavior, or perhaps other aspects of maternal function could potentially affect prenatal and postnatal development [Daston, Developmental toxicology 2 (1994): 189-212].

Examples for effects of maternal toxicity

Developmental stage	Effect of maternal toxicity
Prenatal development	Placenta toxicity
	Maternal nutritional status
	Other nutritional factors
	Gas exchange (oxygen, carbon dioxide carring capacity of blood)
	Stress
	Renal insufficiency (electrolyts homeostasis, removal of metabilic waste)
Postnatal development	Lactation
	Maternal care

Justification for classification or non-classification

It was shown by Seinen, Penninks, Miller, Gründel, Volsen and Kishi (see section immunotoxicity), that the adverse effect belonging to the immne system is an acute effects. Thymus atrophy and depletion of lymph nodes manifested after a one time administration of Dioctyltin to test animals.

Primarily, reproductive toxicity follows the same rules as other types of toxicity and is subject to the same general principles as laid down by Paracelsus in the 16^thCentury “All substances are poisons; there is none which is not a poison. The right dose differentiates a poison from a remedy.’ In addition however, some substances have the potential to selectively affect the developing embryo to cause malformations at dose levels below those causing any other adverse effects.

It is indicated by 67/548/EEC, the ECHA Guidelines and UNECE Guidelines:

“Classification of chemicals as toxic to reproduction is intended to be used for chemicals which have an intrinsic or specific property to produce such toxic effects. Chemicals should not be classified as toxic to reproduction where such effects are solely produced as a non-specific secondary consequence of other toxic effects. Chemicals of most concern are those which are toxic to reproduction at exposure levels which do not produce other signs of toxicity.”

The UNECE discussed in their Final Draft for GHS WEB for Reproductive Toxicity maternal toxicity and criteria therefor:

16. Development of the offspring throughout gestation and during the earlypostnatal stages can be influenced by toxic effects in the mother either through non-specific mechanisms related to stress and the disruption of maternal homeostasis, or by specific maternally mediated mechanisms. So, in the interpretation of the developmental outcome to decide classification for developmental effects it is important to consider the possible influence of maternal toxicity. This is a complex issue because of uncertainties surrounding the relationship between maternal toxicity and developmental outcome. Expert judgment and a weight of evidence approach, using all available studies, should be used to determine the degree of influence that should be attributed to maternal toxicity when interpreting the criteria for classification for developmental effects. The adverse effects in the embryo/fetus should be first considered, and then maternal toxicity, along with any other factors which are likely to have influenced these effects, as weight of evidence, to help reach a conclusion about classification.

 17. Based on pragmatic observation, it is believed, that maternal toxicity may, depending on severity, influence development via non-specific secondary mechanisms, producing effects such as depressed fetal weight, retarded ossification, and possibly resumptions and certain malformations in some strains of certain species. However, the limited numbers of studies which have investigated the relationship between developmental effects and general maternal toxicity have failed to demonstrate a consistent, reproducible relationship across species. Developmental effects which occur even in the presence of maternal toxicity are considered to be evidence of developmental toxicity, unless it can be unequivocally demonstrated on a case by case basis that the developmental effects are secondary to maternal toxicity. Moreover, classification should be considered where there is significant toxic effect in the offspring, e.g. irreversible effects such as structural malformations, embryo/fetal lethality, significant post-natal functional deficiencies.

 18. Classification should not automatically be discounted for chemicals that produce developmental toxicity only in association with maternal toxicity, even if a specific maternally mediated mechanism has been demonstrated. In such a case, classification in Category 2 may be considered more appropriate than Category 1. However, when a chemical is so toxic that maternal death or severe inanition results, or the dams are prostrate and incapable of nursing the pups, it may be reasonable to assume that developmental toxicity is produced solely as a secondary consequence of maternal toxicity and discount the developmental effects. Classification may not necessarily be the outcome in the case of minor developmental changes e.g. small reduction in fetal/pup body weight, retardation of ossification when seen in association with maternal toxicity.

 19. Some of the end points used to assess maternal toxicity are provided below. Data on these end points, if available, needs to be evaluated in light of their statistical or biological significance and dose response relationship.

 Maternal Mortality:  An increased incidence of mortality among the treated dams over the controls should be considered evidence of maternal toxicity if the increase occurs in a dose related manner and can be attributed to the systemic toxicity of the test material. Maternal mortality greater than 10% is considered excessive and the data for that dose level should not normally be considered for further evaluation.

Mating Index(no. animals with seminal plugs or sperm/no. mated x 100)1

Fertility Index(no. animals with implants/no. of matings x 100)1

Gestation Length(if allowed to deliver)

Body Weight and Body Weight Change: Consideration of the maternal body weight change and/or adjusted (corrected) maternal body weight should be included in the evaluation of maternal toxicity whenever such data are available. The calculation ofan adjusted (corrected) mean maternal body weight change, which is the difference between the initial and terminal body weight minus the gravid uterine weight (or alternatively, the sum of the weights of the fetuses), may indicate whether the effect is maternal or intrauterine. In rabbits, the body weight gain may not be useful indicators of maternal toxicity because of normal fluctuations in body weight during pregnancy. Food and Water Consumption (if relevant). The observation of a significant decrease in the average food or water consumption in treated dams compared to the control group may be useful in evaluating maternal toxicity, particularly when the test material is administered in the diet or drinking water. Changes in food or water consumption should be evaluated in conjunction with maternal body weights when determining if the effects noted are reflective of maternal toxicity or more simply, unpalatability of the test material in feed or water.

Clinical evaluations:(including clinical signs, markers, hematology and clinical chemistry studies). The observation of increased incidence of significant clinical signs of toxicity in treated dams relative to the control group may be useful in evaluating maternal toxicity. If this is to be used as the basis for the assessment of maternal toxicity, the types, incidence, degree and duration of clinical signs should be reported in the study. Examples of frank clinical signs of maternal intoxication include: coma, prostration, hyperactivity, loss of righting reflex, ataxia, or labored breathing.

Post-mortem data: Increase incidence and/or severity of post-mortem findings may be indicative of maternal toxicity. This can include gross or microscopic pathological findings or organ weight data, e.g., absolute organ weight, organ-to-body weight ratio, or organ-to-brain weight ratio. When supported by findings of adverse histopathological effects in the affected organ(s), the observation of a significant change in the average weight of suspected target organ(s) of treated dams, compared to those in the control group, may be considered evidence of maternal toxicity.

 

Adverse effects belonging to reproduction are not relevant. 

DOTO-422 -04Animals in the mid dose group show explicit signs of maternal toxicity (thymus atrophie, depletion of lymph nodes) and in the high dose group treatment related increase of unscheduled deaths

DOTAcAc:

Adverse effects belonging to reproduction are not relevant. Animals in the low dose group show explicit signs of maternal toxicity (thymus atrophie, depletion of lymph nodes) and in the mid dose group treatment related increase of unscheduled deaths

In accordance with the ECHA - , OECD -, UN – Guidelines and with 67/548/EEC the NOEAL for Diocthyltin oxide is set to 5 mg /kg diet.

A NOAEL for reproductive toxicity was not considered indicated because the reproductive effects observed were non-specific and considered to be secondary to maternal toxicity

Based on the effects noted in the thymus in both male and female , the NOAEL (Study DOTO 422 -04) was concluded to be the lowest group tested, 5 mg/kg diet which was equivalent to 0.3-0.4 mg/kg bw/day for male animals and 0.3-0.5 mg/kg bw/day for female animals.

There are no relevant adverse findings concerning development and reproduction.