Ethyl 9,9-dioctyl-4,7,11-trioxo-3,8,10-trioxa-9-stannatetradeca-5,12-dien-14-oate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Extended one-generation reproductive toxicity study (EOGRTS) (Annex IX, Section 8.7.3; test method: OECD TG 443) in rats:

This information will be submitted later based on ECHA decision number CCH-D-2114461461-54-01/F.

Please refer to the letter submitted on behalf of Galata Chemicals GmbH and the OrganoTin REACH Consortium dated 12 November 2020 for the full details of the testing.

The dose range-finding study was initiated in early June 2021, but ran into technical problems and the study had to be terminated due to severe toxicity observed in all test item groups, consisting of severe body weight loss and extremely low food consumption. It was determined that the available data used to set the dose levels (performed at a different lab) had not translated. A repeat dose range-finding study was initiated early September and the in-life phase of the study should finish late November 2021 and the draft report is due to be issued 11 February 2022.

Due to the extremely long lead in time for the EOGRTS, the requirement to repeat the dose range-finding study has fortunately not impacted on the planned schedule for the study. The EOGRTS is planned to commence mid to late March 2022 and assuming the F2-generation is not triggered will finish early October 2022, with the draft report due to be issued late February 2023.

As soon as all the final reports are available (and it should be noted that it is likely, due to the nature of the studies, it could easily take 2 to 3 months for them to be reviewed and finalised, possibly longer depending upon the findings) then the data will be incorporated into the registration dossier and the CSR revised and an update will be submitted to ECHA. Overall based on the current study schedules we would anticipate that the registration including all the new data required to meet the Decision on a compliance check will be submitted Q3 2023 (this could be extended if the F-2 generation is triggered on the EOGRTS).

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

The target substance is a mono-constituent organotin substance that consists of a tin as central metal element with two octyl-ligands. The source substance Dioctyltin oxide (DOTO) (EC Number 212-791-1 and CAS 870-08-6) is also an organotin compound that has the identical structure elements as the target substance in respect of the tin-alkyl moiety.
According to WHO IPCS CIRCAD (2006) organotin compounds are characterized by a tin–carbon bond and have the general formula RxSn(L)(4−x), where R is an organic alkyl or aryl group and L is an organic (or sometimes inorganic) ligand. The organotin moiety is significant toxicologically. The anionic ligand influences physicochemical properties but generally has little or no effect on the toxicology.
Since the target substance and the source substances share the identical organotin moiety, and the organotin moiety is generally recognized as the relevant toxophore of organotins and the toxicity estimates (AE) respectively toxicity limits for organotins are expressed as tin, the overall ecotoxicity/systemic toxicity of the target can be interpolated by assessing the (eco-)toxicity of the source (WHO IPCS CIRCAD, 2006, BAUA AGS TRGS 900, 2014, Summer KH, Klein D and Greim H, 2003).
The purity of the source and target substance are expected to be similar, based on the manufacturing method. The impurity profile is not expected to have strong effects on substance properties and any impurity of (eco-)toxicological relevance of the source substances is expected to be present in the target substance. Consequently, the hazard profiles of the source substances, including those of their impurities, are intrinsically covered. Differences in impurities are not expected and thus do not have an impact on the (eco-)toxic properties.

References
BAUA (Bundesanstalt für Arbeitsschutz und Arbeitsmedizin (Federal Institute for Occupational Safety and Health)) AGS (Ausschuss für Gefahrstoffe (Committee on Hazardous Substances)) TRGS (Technical Rules for Hazardous Substances) 900 (2014). Begründung zu n-Octylzinnverbindungen, April 2014.
Summer KH, Klein D, Griem H (2003). Ecological and toxicological aspects of mono- and disubstituted methyl-, butyl-, octyl-, and dodecyltin compounds - Update 2002. GSF National Research Center for Environment and Health, Neuherberg, for the Organotin Environmental Programme (ORTEP) Association.
World Health Organization (WHO) International Programme on Chemical Safety (IPCS) Concise International Chemical Assessment Document (CICAD) 73 Mono- and disubstituted methyltin, butyltin, and octyltin compounds (2006). Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organization, and the World Health Organization, and produced within the framework of the Inter-Organization Programme for the Sound Management of Chemicals.

Reason / purpose for cross-reference:

read-across source

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

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.:

25 mg/kg diet

System:

immune system

Organ:

thymus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

250 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No primary effects on reproduction

Critical effects observed:

no

Reproductive effects observed:

not specified

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key 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.

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

Dose / conc.:

0 mg/kg diet

Remarks:

Range finding study

Dose / conc.:

25 mg/kg diet

Remarks:

Range finding study

Dose / conc.:

75 mg/kg diet

Remarks:

Range finding study

Dose / conc.:

200 mg/kg diet

Remarks:

Range finding study

Dose / conc.:

500 mg/kg diet

Remarks:

Range finding study

Dose / conc.:

0 mg/kg diet

Remarks:

Main study

Dose / conc.:

5 mg/kg diet

Remarks:

Main study

Dose / conc.:

25 mg/kg diet

Remarks:

Main study

Dose / conc.:

250 mg/kg diet

Remarks:

Main study

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

Oestrous cyclicity (parental animals):

-

Sperm parameters (parental animals):

-

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

Description (incidence and severity):

In the 25 mg/kg diet grou there were adverse finding in the thymus

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:

no effects observed

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

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:

no effects observed

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

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg diet

System:

immune system

Organ:

thymus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

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.

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

250 mg/kg diet

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no primary effects on reproduction

Key result

Critical effects observed:

no

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 for general toxicity 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.

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.

Reference 3

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

This information will be submitted later based on ECHA decision number CCH-D-2114461461-54-01/F.
Please refer to the letter submitted on behalf of Galata Chemicals GmbH and the OrganoTin REACH Consortium dated 12 November 2020 for the full details of the testing.
Extended one-generation reproductive toxicity study (EOGRTS) (Annex IX, Section 8.7.3; test method: OECD TG 443) in rats: The dose range-finding study was initiated in early June 2021, but ran into technical problems and the study had to be terminated due to severe toxicity observed in all test item groups, consisting of severe body weight loss and extremely low food consumption. It was determined that the available data used to set the dose levels (performed at a different lab) had not translated. A repeat dose range-finding study was initiated early September and the in-life phase of the study should finish late November 2021 and the draft report is due to be issued 11 February 2022.
Due to the extremely long lead in time for the EOGRTS, the requirement to repeat the dose range-finding study has fortunately not impacted on the planned schedule for the study. The EOGRTS is planned to commence mid to late March 2022 and assuming the F2-generation is not triggered will finish early October 2022, with the draft report due to be issued late February 2023.
As soon as all the final reports are available (and it should be noted that it is likely, due to the nature of the studies, it could easily take 2 to 3 months for them to be reviewed and finalised, possibly longer depending upon the findings) then the data will be incorporated into the registration dossier and the CSR revised and an update will be submitted to ECHA. Overall based on the current study schedules we would anticipate that the registration including all the new data required to meet the Decision on a compliance check will be submitted Q3 2023 (this could be extended if the F-2 generation is triggered on the EOGRTS).

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1.4 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

As the study was performed on a read-across substance, the study was assigned a reliability score of 2 (reliable with restrictions) in accordance with the criteria for assessing data quality as outlined in Klimisch (1997) and considered suitable for assessment as an accurate reflection of the test material.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The toxicological effect of the test material 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. Only one death was noted during the study, and this was not attributed to toxicity of the test material.

Based on the effects noted in the thymus in both male and female rats 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 0.3-0.4 mg/kg bw/day for male animals and 0.3-0.5 mg/kg bw/day for female animals.

Based on effects on reproductive parameters (mean duration of gestation, post-implantation loss, total number of still born pups) the NOAEL for reproductive toxicity was determined to be 25 mg/kg diet, equivalent to 1.5-1.7 mg/kg bw/day in males and 1.4-2.4 mg/kg bw/day in female animals. All these non-specific effects are considered likely to be related to maternal toxicity (severe effects on body weight/body weight gain and food consumption during pregnancy and lactation; thymus effects).

Short description of key information:
NOAEL (reproduction) = 25 mg/kg diet in rats, equivalent to 1.5-1.7 mg/kg bw/day in males and 1.4-2.4 mg/kg bw/day in female rats (based on effects on reproductive/developmental parameters), OECD 422, Waalkens-Berendsen 2004.

Justification for selection of Effect on fertility via oral route:
Only one study was available for evaluation. The presented key study was performed in line with a standardised guideline and performed to GLP. The level of reporting was to a high standard, sufficient to accurately assess the quality of the data presented. Due to the nature of the effects observed, and as an effect level could be derived from the results of the study sufficient to be carried forward to risk assessment, the key study was therefore considered to be an accurate account of the substance's effects on fertility.

Effects on developmental toxicity

Description of key information

This information will be submitted later based on ECHA decision number CCH-D-2114461461-54-01/F.
Please refer to the letter submitted on behalf of Galata Chemicals GmbH and the OrganoTin REACH Consortium dated 12 November 2020 for the full details of the testing.
Pre-natal developmental toxicity study (Annex IX, Section 8.7.2.; test method: OECD TG 414) in a first species (rat): Development and validation of an analytical method for the in vivo toxicity studies including trial formulation and trial diet analysis was started March 2021 and this proved technically challenging taking account the properties of the substance, but it was successfully completed at the end of July 2021.
The main developmental study was then initiated early October 2021, but the study soon ran into technical problems, as the top and middle doses that had been set based on the range-finding study performed at a different lab were found not to translate and these groups had to be to be sacrificed on ethical grounds due to excessive maternal toxicity. The low dose was able to be continued to completion and will now be the top dose for the study, but two additional levels need to be run to ensure a guideline compliant study. These additional dose levels are scheduled to commence on the 10 Jan 2022 and the in-life phase of the study should finish the 28 January 2022, with the draft report due to be issued the 08 April 2022.
As soon as all the final reports are available (and it should be noted that it is likely, due to the nature of the studies, it could easily take 2 to 3 months for them to be reviewed and finalised, possibly longer depending upon the findings) then the data will be incorporated into the registration dossier and the CSR revised and an update will be submitted to ECHA. Overall based on the current study schedules we would anticipate that the registration including all the new data required to meet the Decision on a compliance check will be submitted Q3 2023 (this could be extended if the F-2 generation is triggered on the EOGRTS).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

This information will be submitted later based on ECHA decision number CCH-D-2114461461-54-01/F.
Please refer to the letter submitted on behalf of Galata Chemicals GmbH and the OrganoTin REACH Consortium dated 12 November 2020 for the full details of the testing.
Pre-natal developmental toxicity study (Annex IX, Section 8.7.2.; test method: OECD TG 414) in a first species (rat): Development and validation of an analytical method for the in vivo toxicity studies including trial formulation and trial diet analysis was started March 2021 and this proved technically challenging taking account the properties of the substance, but it was successfully completed at the end of July 2021.
The main developmental study was then initiated early October 2021, but the study soon ran into technical problems, as the top and middle doses that had been set based on the range-finding study performed at a different lab were found not to translate and these groups had to be to be sacrificed on ethical grounds due to excessive maternal toxicity. The low dose was able to be continued to completion and will now be the top dose for the study, but two additional levels need to be run to ensure a guideline compliant study. These additional dose levels are scheduled to commence on the 10 Jan 2022 and the in-life phase of the study should finish the 28 January 2022, with the draft report due to be issued the 08 April 2022.
As soon as all the final reports are available (and it should be noted that it is likely, due to the nature of the studies, it could easily take 2 to 3 months for them to be reviewed and finalised, possibly longer depending upon the findings) then the data will be incorporated into the registration dossier and the CSR revised and an update will be submitted to ECHA. Overall based on the current study schedules we would anticipate that the registration including all the new data required to meet the Decision on a compliance check will be submitted Q3 2023 (this could be extended if the F-2 generation is triggered on the EOGRTS).

Species:

rat

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 2

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

The target substance is a mono-constituent organotin substance that consists of a tin as central metal element with two octyl-ligands. The source substance Dioctyltin oxide (DOTO) (EC Number 212-791-1 and CAS 870-08-6) is also an organotin compound that has the identical structure elements as the target substance in respect of the tin-alkyl moiety.
According to WHO IPCS CIRCAD (2006) organotin compounds are characterized by a tin–carbon bond and have the general formula RxSn(L)(4−x), where R is an organic alkyl or aryl group and L is an organic (or sometimes inorganic) ligand. The organotin moiety is significant toxicologically. The anionic ligand influences physicochemical properties but generally has little or no effect on the toxicology.
Since the target substance and the source substances share the identical organotin moiety, and the organotin moiety is generally recognized as the relevant toxophore of organotins and the toxicity estimates (AE) respectively toxicity limits for organotins are expressed as tin, the overall ecotoxicity/systemic toxicity of the target can be interpolated by assessing the (eco-)toxicity of the source (WHO IPCS CIRCAD, 2006, BAUA AGS TRGS 900, 2014, Summer KH, Klein D and Greim H, 2003).
The purity of the source and target substance are expected to be similar, based on the manufacturing method. The impurity profile is not expected to have strong effects on substance properties and any impurity of (eco-)toxicological relevance of the source substances is expected to be present in the target substance. Consequently, the hazard profiles of the source substances, including those of their impurities, are intrinsically covered. Differences in impurities are not expected and thus do not have an impact on the (eco-)toxic properties.

References
BAUA (Bundesanstalt für Arbeitsschutz und Arbeitsmedizin (Federal Institute for Occupational Safety and Health)) AGS (Ausschuss für Gefahrstoffe (Committee on Hazardous Substances)) TRGS (Technical Rules for Hazardous Substances) 900 (2014). Begründung zu n-Octylzinnverbindungen, April 2014.
Summer KH, Klein D, Griem H (2003). Ecological and toxicological aspects of mono- and disubstituted methyl-, butyl-, octyl-, and dodecyltin compounds - Update 2002. GSF National Research Center for Environment and Health, Neuherberg, for the Organotin Environmental Programme (ORTEP) Association.
World Health Organization (WHO) International Programme on Chemical Safety (IPCS) Concise International Chemical Assessment Document (CICAD) 73 Mono- and disubstituted methyltin, butyltin, and octyltin compounds (2006). Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organization, and the World Health Organization, and produced within the framework of the Inter-Organization Programme for the Sound Management of Chemicals.

Reason / purpose for cross-reference:

read-across source

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

Abnormalities:

no effects observed

Dose descriptor:

NOAEC

Effect level:

> 25 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Viability index day 1-4

Remarks on result:

other: effects only with high maternal toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key 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.

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Main study

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

Main study

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Main study

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Main study

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Range finding test

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

Range finding test

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Range finding test

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Range finding test

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Range finding test

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

adverse effects in the immune system (thymus)

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):

not specified

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

Description (incidence and severity):

thymus

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increase in liver weight, decrease of thymus weight

Gross pathological findings:

not specified

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not specified

Early or late resorptions:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

only with high maternal toxicity

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not specified

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.

Key result

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

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

only with high maternal toxicity

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

only with high maternal toxicity

External malformations:

not examined

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

not specified

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.

Key result

Dose descriptor:

NOAEC

Effect level:

> 25 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Viability index day 1-4

Remarks on result:

other: effects only with high maternal toxicity

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 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.

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1.4 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

As the study was performed on a read-across substance, the study was assigned a reliability score of 2 (reliable with restrictions) in accordance with the criteria for assessing data quality as outlined in Klimisch (1997) and considered suitable for assessment as an accurate reflection of the test material.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The developmental effect of the test material 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, and this was not attributed to toxicity of the test material.

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.

Based on post-implantation loss, total number of stillborn pups, pup mortality on PN 4, pup weight on PN 1 and the number of runts, the NOAEL for developmental toxicity was determined to be 25 mg/kg diet. All these non-specific effects are considered likely to be related to maternal toxicity (severe effects on body weight/body weight gain and food consumption during pregnancy and lactation; thymus effects).

Justification for selection of Effect on developmental toxicity: via oral route:
Only one study was available for evaluation. The presented key study was performed in line with a standardised guideline and performed to GLP. The level of reporting was to a high standard, sufficient to accurately assess the quality of the data presented. Due to the nature of the effects observed, and as an effect level could be derived from the results of the study sufficient to be carried forward to risk assessment, the key study was therefore considered to be an accurate account of the substance's effects on development.

Justification for classification or non-classification

In accordance with the Regulation EC No. 1272/2008 on a precautionary basis the substance is classified as Category 2: H361 Suspected of damaging fertility or the unborn child, although the non-specific effects observed on reproductive/developmental parameters (mean duration of gestation, post-implantation loss, total number of still born pups, pup mortality to PN day 4, pup weight on PN day 1 and number of runts) are likely to be related to maternal toxicity (severe effects on body weight/body weight gain and food consumption during pregnancy and lactation; thymus effects) as evidenced by the NOAEL for systemic toxicity, lower than that for reproductive/developmental toxicity, obtained in this same study.