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Administrative data

Description of key information

No reliable study with single superphosphate is present.

However, an expert statement shows no adverse effects are expected for SSP, considering both the values derived for safe use of the relevant ions and the results of a reliable OECD 422 study with the substance’s analogue TSP.

The read-across rationale and the expert statement are included in both IUCLID and the CSR.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 December 2001 - 27 May 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 22.03.96
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 297 to 365 g for males and 194 to 251 g for females
- Fasting period before study: not applicable
- Housing:Unless paired for mating, the animals were singly housed in RB3 modified cages consisiting of high density polypropylene bodies with lids and floors of stainless steel grid. These cages were suspended in batteries over trays lined with absorbent paper. RB3 modified cages were used
throughout the study with the exception of reproductive subgroup females from Day 17 after mating, where RB3 solid-bottomed cages were used.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-70%, , on one occasion during the acclimatization period relative humidity was recorded as 20% and was re-established within
24 hours.
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 December 2001 To: 19 February 2002
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The formulation for Group 4 (150 mg/mL) was mixed by adding the required volume of vehicle (water purified by reverse osmosis) to the required
weight of GTSP and magnetically stirring for up to 1 hour until a visibly homogenous brown suspension was formed. Formulation for Groups 3 and 2
were prepared by direct dilution of this suspension.

VEHICLE
- Concentration in vehicle: 25 - 75 - 150 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration during Weeks 1,4 and 6 of the study were analysed using spectrophotometry for test
material content and found to be satisfactory.
Duration of treatment / exposure:
Animals were divided between two subgroups (toxicity and reproductive subgroups).
Males of both subgroups and females of the toxicity subgroup were treated until termination during week 6 of treatment. Doses were administered to the reproductive subgroup females for two weeks prior to pairing, throughout pairing and gestation until Day 3 of lactation. Animals that were
in parturition at the time of dosing were not dosed that day. Control animals received the vehicle over the same treatment period.
Animals were not dosed on their scheduled day of necropsy.
Frequency of treatment:
Daily
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
The toxicity subgroup consisted of 5 males and 5 females per group and the reproductive subgroup consisted of 10 females and
5 males per group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: As no treatment-related effects were seen on parameters assessed in a preliminary study at 1000 mg/kg/day, the high
dosage of 1500 mg/kg/day was selected by the Sponsor to attempt to elicit an overt toxic response and establish a reference level for toxicity.
The concept of a 'Limit Dose' is accepted by the OECD and is generally set in the region of 1000 mg/kg/day; therefore, it was not considered
necessary to investigate a dosage above 1500 mg/kg/day.

- Rationale for animal assignment (if not random): The animal numbers were assigned to groups in non-sequential manner due to the need for the
functional observation battery and detailed clinical signs to be performed without knowledge of the treatment groups to which animals were assigned.

- Section schedule rationale (if not random): The sequence in which the animals of the toxicity subgroup were killed after completion of the
observation period was selected to allow satisfactory inter-group comparison. Reproductive subgroup males were killed after the toxicity subgroup
animals and reproductive subgroup females were killed on Day 4 of lactation.
Positive control:
No.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- Cage side observations: evidence of reaction to treatment or ill-health

DETAILED PHYSICAL OBSERVATIONS: Yes
- Time schedule: weekly
- A more detailed physical examination was performed on each animal by an observer. After removal from the home cage, animals were
assessed for physical condition and behaviour during handling and after being placed in a standard arena. During these examinations particular
attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behaviour.

ADDITIONAL DETAILED OBSERVATIONS: Yes
- Time schedule: during Week 1 daily according to the following frequency:
1. Pre-dose observation, 2.As each animal was returned to its home cage, 3.At the end of dosing each group, 4.Between 1 and 2 hours after
completion of dosing all groups and 5.As late as possible in the working day.
From Week 2 to termination daily observations were limited to 1 and 4.

BODY WEIGHT: Yes
- Time schedule for examinations: Each male and toxicity subgroup female: on the day that treatment commenced, weekly thereafter, and at necropsy. Reproductive subgroup females: on the first day of treatment, weekly until pairing and on Days 0, 7, 14, 17 and 20 after mating, and Days 1 and 4 of
lactation, and at necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for observations: All males and toxicity subgroup females: weekly throughout the study with the exception of the males whilst in
pairing with the reproductive subgroup females. Reproductive subgroup females: weekly before pairing then on Days 0,7,14, 17 and 20 after mating
and Days 1 and 4 of lactation. No food consumption was recorded for toxicity subgroup males or reproductive subgroup animals during pairing.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight
gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During week 5, following FOB examinations
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: toxicity subgroup animals (5 males + 5 females per dose group)
- Parameters checked: Haematocrit (Hct), Haemoglobin (Hb), Red blood cell count (RBC), Mean cell haemoglobin (MCH), Mean cell haemoglobin
concentration (MCHC), Mean cell volume (MCV), Total white cell count (WBC), Differential WBC count, Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Pit), Reticulocyte count (Retic), Blood film for abnormal morphology and
unusual cell types, including normoblasts (The most common morphological changes, anisocytosis (aniscyto), micro/macrocytosis (Microcyto/
Macrocyto), hypo/hyperchromasia (Hypochrom/Hyperchrom), Platelet clumping (PIt. Clump)), Prothrombin time (PT) and Activated partial
thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During week 5, following FOB examinations
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: toxicity subgroup animals (5 males + 5 females per dose group)
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST),
Gamma-glutamyl transpeptidase (gGT), Total Bilirubin (Bili), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Sodium (Na),
Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic Phosphorus (Phos), Magnesium (Mg), Total protein (Total Prot), Albumin (Alb),
Albumin/globulin ratio (A/G Ratio)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: After 4 weeks of treatment
- Dose groups that were examined: Toxicity subgroup animals (5 males + 5 females per dose group)
- Battery of functions tested: Approach response, Touch response, Startle reflex (auditory), Tail pinch response, Grip strength: forelimb and
hindlimb and motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, a detailed examination of the external features and orifices, the neck and associated tissues and the cranial, thoracic,
abdominal and pelvic cavities and their viscera.

Toxicity subgroup animals:
Animals were killed during Week 6 of treatment by carbon dioxide inhalation and subjected to a detailed necropsy.
The requisite organs were weighed after being dissected free of adjacent fat and other contiguous tissue. External and cut surfaces of the organs and tissues were examined as appropriate, abnormalities and interactions were noted and the required tissue samples and any abnormalities
preserved in appropriate fixative.
The following tissues were retained: Adrenals (left and right)*, Aorta - thoracic, Brain*, Caecum, Colon, Duodenum, Epididymides (left and right)*,
Eyes, Heart*, Ileum, Jejunum, Kidneys (left and right)*, Liver*, Lungs (including bronchi), Lymph nodes - mandibular, Lymph nodes - mesenteric,
Lymph nodes - regional to masses, Mammary area, Oesophagus, Optic nerves, Ovaries (left and right)*, Pancreas, Pituitary*, Prostate *, Rectum,
Salivary glands, Sciatic nerves, Seminal vesicles*, Skin, Spinal cord, Spleen*, Sternum, Stomach, Testes (left and right)*, Thymus*, Thyroids with
parathyroids**, Trachea, Urinary bladder, Uterus with cervix*, Vagina
* Organ weight recorded / * * Organ weight recorded after fixation.

Reproductive subgroup animals:
Males were killed after the toxicity subgroup animals. They were subjected to a detai led necropsy for evidence of disease or adverse reaction to
treatment. The testes, epididymides, seminal vesicles with coagulating gland and prostate were retained for each animal. Females were killed on
Day 4 of lactation by carbon dioxide inhalation and subjected to a detailed necropsy for evidence of disease or adverse reaction to treatment.
The number of implantation sites was recorded and the ovaries, uterus with oviducts and cervix, vagina, pituitary and mammary tissue retained in appropriate fixative. Abnormal tissues were also retained in appropriate fixative. Offspring of the reproductive subgroup females were killed by
intraperitoneal injection of sodium pentobarbitone on Day 4 of age and subjected to a macroscopic necropsy. Abnormal tissues were also retained in appropriate fixative. Any offspring dying before scheduled termination were subjected to necropsy and assessment of the stomach for milk
content.

HISTOPATHOLOGY: Yes

Toxicity subgroup animals:
Organs (including abnormalities) preserved at macroscopic necropsy were processed for animals from the Control and high dosage groups
(Groups 1 and 4). Following treatment related findings in Group 4, the stomachs and kidneys of intermediate and low dosage animals were also
processed and examined. Tissue samples were dehydrated, embedded in paraffin wax and sectioned at approximately four to five micron thickness. Staining was with haematoxylin and eosin, except testes, which were stained with periodic acid/schiff (PAS).
The following tissues were scheduled for histopathology: Abnormalities, Adrenals (cortex and medulla), Aorta (thoracic), Brain (cerebrum,
cerebellum, midbrain), Caecum, Colon, Duodenum, Epididymides (longitudinal section to include the caput and cauda epididymides), Eyes
(longitudinal section of each (both examined)), Heart (including auricular and ventricular regions), Ileum, Jejunum, Kidneys (including cortex,
medulla and papilla regions), Liver (section from all main lobes), Lungs (section from two major lobes, to include bronchi), Lymph nodes,
Mammary area, Oesophagus, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands, Sciatic nerves (only one examined), Seminal vesicles,
Skin, Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), Spleen, Sternum (with bone marrow),
Stomach (keratinised, glandular and antrum), Tissue (region to be examined (where appropriate)), Testes, Thymus, Thyroid (included parathyroid
in section, where possible), Trachea, Urinary bladder, Uterus (uterus section separate from cervix section (both examined)), Vagina.

For bilateral organs sections of both the left and right organs were examined. A separate section was prepared from each of the remaining tissues.

In addition to the general histopathological examination of all testicular elements, including tubular and interstitial compartments,
spermatogenesis was assessed taking into consideration the stages of the spermatogenic cycle. Where no disturbances were detected, the testes
were recorded as normal.

Reproductive subgroup animals:
Histology for reproductive subgroup animals was restricted to the retained reproductive organs and abnormalities observed at macroscopic
necropsy.
Other examinations:
Mating procedure:
All 10 males in each group (toxicity and reproductive subgroups) were mated with the 10 reproductive subgroup females after all animals had
received 2 weeks of treatment. Pairing was on a one-to-one basis within treatment groups for up to 2 weeks, although all animals mated and were
separated within 1 week. Each morning, following pairing, the trays beneath the cages were checked for ejected copulation plugs and a wet vaginal
smear was prepared from each female and examined for the presence of spermatozoa. The day on which a sperm positive vaginal smear or at least
three copulation plugs were found was designated Day 0 of gestation. Once mating had been confirmed, males and females were separated and the
males were returned to their normal group housing.

Parturition observations and gestation length:
From Day 20 after mating reproductive subgroup animals were checked 3 times daily for evidence of parturition. The females were permitted to
deliver their young naturally and rear their own offspring until Day 4 of lactation. Numbers of live and dead offspring were recorded during the
parturition process.

Offspring observations:
All offspring were examined at approximately 24 hours after birth (Day 1) and the number of offspring born (live and dead) was recorded.
Live offspring were individually identified within each litter by toe tattoo and any clinical observations were recorded.
Litters were observed daily for evidence of abnormal appearance or behaviour. Daily records were maintained of mortality and consequent
changes in litter size. Where practical, any offspring found dead were subjected to a macroscopic examination as soon as possible.
The offspring were sexed at individual weighing on Day 1 and Day 4 of age.
Statistics:
For some parameters, the similarity of the data was such that analyses were not considered to be necessary.
For categorical data, the proportion of animals was analysed using Fisher's Exact test.
For continuous data, Bartlett's test was first applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome
of Bartlett's test, treated groups were then compared with the Control group, incorporating adjustment for multiple comparisons where necessary.
For bodyweight gains and organ weights, whenever Bartlett's test was found to be statistically significant, a Behrens-Fisher test was used to perform
pairwise comparisons, otherwise a Dunnett's test was used.
The following sequence of statistical tests was used for grip strength and motor activity, bodyweight change during gestation and lactation,
bodyweight and bodyweight change for offspring, food consumption and clinical pathology data:
- If 75% of the data were the same value, then a frequency analysis was applied. Treatment groups were compared using a Mantel test for a trend in
proportions and also pairwise Fisher's Exact tests for each dose group against the control.
- If Bartlett's test for variance homogeneity was not significant at the 1 % level, then parametric analysis was applied. If the F 1 test for monotonicity of dose-response was not significant at the 1 % level, Williams' test for a monotonic trend was applied. If the F 1 test was significant, Dunnett's test was
performed instead.
- If Bartlett's test was significant at the 1 % level, then logarithmic and square-root transformations were tried. If Bartlett's test was still significant, the non-parametric tests were applied. If the HI test for monotonicity of dose-response was not significant at the 1 % level, Shirley's test for a monotonic
trend was applied. If the HI test was significant, Steel's test was performed instead.

Significant differences were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: One control female was found dead with findings that were consistent with a dosing error. This death was therefore considered to be unrelated to the test material.
A dosage-dependant increase in transient post-dosing salivation was apparent, and this was considered more likely to be due to the palatability of
the test formulations than to an effect of the test material.

BODY WEIGHT AND WEIGHT GAIN:
Body weight gain for toxicity subgroup females at 1500 mg/kg/day appeared to be suppressed during weeks 1-2 of the study. Overall body weight
gain for all treated reproductive subgroup females between weeks 0-2 was slightly reduced compared to control values.
Body weight gain appeared to be unaffected for males during the entire study and for females after week 2.

FOOD CONSUMPTION:
Food consumption of all treated reproductive subgroup females appeared to be lower when compared to Control during week 2 and during the first
week of gestation. Food consumption of reproductive subgroup females at 750 and 1500 mg/kg was slightly lower than Control during the first
4 days of lactation. There were no effects of treatment on the amount of food consumed by toxicity subgroup males and females and by reproductive subgroup males. The difference between toxicity and reproductive subgroup females may be due to different diets administered to the subgroups.

HAEMATOLOGY:
Although neutrophil counts in males and females at 1500 mg/kg were variable, they were generally elevated, possibly reflective of the inflammatory
response in the stomach. Marginally increased platelet counts and a reduction in activated partial thromboplastin time were observed for males at
1500 mg/kg/day. Females at 1500 mg/kg showed increased platelet and basophil counts and a decrease in activated partial thromboplastin time.
One female at 750 mg/kg and one female at 1500 mg/kg showed increased white blood cell counts.

CLINICAL CHEMISTRY:
The following changes were recorded for which an effect of treatment could not be discounted:
lower phosphorous levels at 1500 mg/kg; lower total protein with elevated albumin/globulin ratios at 750 and 1500 mg/kg; lower albumin levels in all treated groups (males only); a non dosage-dependant slight reduction of calcium levels at 750 and 1500 mg/kg (males only) and lower total bilirubin levels at 1500 mg/kg (females only).

NEUROBEHAVIOUR:
Although motor activity data were highly variable, there appeared to be a general reduction in both ambulatory and rearing activity for males at 1500 mg/kg. The fore-limb strength of males at 1500 mg/kg appeared to be reduced but not statistically significant and in the absence of an effect of hind-limb grip strength. No effects were observed for females.

GROSS PATHOLOGY:
A number of toxicity subgroup males and females at 750 and 1500 mg/kg showed stomach findings (including thickened and dark areas) and
horizontal bands on the incisors. One male at 250 mg/kg showed also horizontal bands on the incisors.

HISTOPATHOLOGY: NON-NEOPLASTIC
Basophilia in the cortical tubules of the kidneys were observed in the majority of males of the toxicity subgroups (0/5, 3/5, 4/5 and 4/5 at dose levels of 0, 250, 750 and 1500 mg/kg, respectively) and one female in each of the treated toxicity subgroups. According to the authors: Although this is not an uncommon finding, due to the high preponderance in male treated groups and no occurrences among control animals, it was considered related to treatment. However the reviewer does not agree: all findings were minimal except for one male in the highest dose group (slight) and minimal to slight degrees of tubular basophilila in kidneys may be encountered in kidneys of untreated rats. also in both severity and incidence no dose response can be found. At greater severity, it is a characteristic of induced nephropathies. Therefore this is not considered adverse. References: Gopinath et al. 1987: Atlas of experimental toxicological pathology, and Hard et al. 1999: Non-proliferative lesions of the kidney and lower urinary tract in the rat, URG-1.
Stomachs of toxicity subgroup animals showed (mostly) minimal or slight degenerative/inflammatory changes at all doses but not in control animals. It is thought that these findings may have been associated with an irritant effect of the test formulations rather than systemic toxicity.
Histological processing of the teeth that showed horizontal banding failed to detect any involvement of areas examined suggesting that the banding was probably restricted to the enamel of the teeth. This directs to a mineralisation process of tooth and bones.

OTHER FINDINGS:
Group mean body weights of offspring at Day 1 of age through to Day 4 were slightly lower than control, and both sexes showed a similar response (statistically significant for females). This may suggest in utero growth retardation and a relation to treatment cannot be discounted.
The following parameters were unaffected by treatment: pre-coital interval, mating performance and fertility, gestation length and gestation index, litter size, offspring survival indices and sex ratio.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
250 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: general toxicity: haematological and clinical parameters and horizontal banding of the teeth.
Critical effects observed:
not specified
Conclusions:
NOAEL(systemic) = 250 mg/kg bw/d for general toxicity based on haematological and clinical parameters and horizontal bands on incisors
NOAEL (local) <250 mg/kg bw/d for general toxicity based on irritating effects in the stomach
NOAEL: 750 mg/kg/day for reproduction/developmental toxicity based on body weights of offspring.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles. However, since the study was performed with a substance analogue and the data are read across, the Klimisch score is 2.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: oral

No reliable study with single superphosphate is present. In a reliable oral OECD 422 study, rats (5/sex/dose) were dosed 250, 750 and 1500 mg/kg bw/day triple superphoshate. Males and females were treated until termination during week 6 of treatment. The systemic NOAEL was determined to be 250 mg/kg bw/d. The critical effect at 750 mg/kg bw/d was the horizontal banding of the teeth, which directs to a mineralisation process of tooth and bones. Histological processing of the teeth that showed horizontal banding failed to detect any involvement of areas examined suggesting that the banding was probably restricted to the enamel of the teeth.

Based on an expert statement, no further studies are considered necessary. Considering the values derived for safe use of the relevant ions and the data on its analogue TSP it is highly unlikely that SSP will show any effects in man. Exposure to the ions from uses regulated under REACH is considered to be negligible compared to the natural abundancy in humans of calcium, phosphate and sulphate.


Repeated dose: dermal/inhalation

No dermal or inhalation toxicity studies are present. Although dermal exposure is likely, dermal absorption is considered to be very low and thus a dermal exposure study would be of limited value. In addition,the vapour pressure is very low and the particle size of the substance has an MMAD of > 200μm, making the possible inhalation exposure negligible (see particle size distribution results), indicating that only very limited inhalation exposure is possible.

Justification for classification or non-classification

Based on the available data, single superphosphate does not have to be classified according to the CLP Regulation for repeated dose toxicity.