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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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:
18 January 2010 - 16 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD guideline 422 and under GLP conditions.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Phosphorous slag
- Physical state: Solid
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbrescle Cedex, France
- Age at study initiation: (P) approx. 11 wks
- Mean weight at study initiation: (P) Males: 330-338 g; Females: 197-199 g
- Housing:
Pre-mating: groups of 5/sex/cage
Mating: 1:1
Post-mating: males in groups of 5, females individually

- Diet: Ad libitum, pelleted rodent diet
- Water: Ad libitum, tap-water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8-21.7
- Humidity (%): 21-69
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous carboxymethyl cellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations prepared daily within 6 hours prior to dosing and homogenized to a visually acceptable level.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing facility
- Amount of vehicle (if gavage): 5 ml/kg bw
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No chemical analyses conducted, since no analytical method could be developed as the substance consisted of numerous components
Duration of treatment / exposure:
Males: 29 days (2 weeks prior to mating, during mating and up to termination)
Females: 43-56 days (2 weeks prior to mating, during mating, during pregnancy and at least 4 days of lactation)
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based on results of a 10-day dose range finding study
- Rationale for animal assignment: Random according to body weight (with all animals within +/- 20% of the sex mean)
Positive control:
Not relevant

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily
- Cage side observations: Mortality, viability

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Weekly

BODY WEIGHT:
- Time schedule for examinations: Weekly throughout the study and more frequent in females during preganancy (day 0, 4, 7, 11, 14, 17 and 20) and lactation (day 1 and 4)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption: Recorded weekly, except for males and females which were housed together for mating. More frequent examination in females during preganancy (day 0, 4, 7, 11, 14, 17 and 20) and lactation (day 1 and 4)

WATER CONSUMPTION):
By subjective appraisal

HAEMATOLOGY:
- Time schedule for collection of blood: Prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Iso-flurane
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked: WBC, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Prior to scheduled post mortem examination
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked: ALAT, ASAT, ALP, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate, bile acids

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: Males during week 4 of treatement, females during lactation
- Dose groups that were examined: 5 animals/sex/group
- Battery of functions tested: hearing ability / pupillary reflex / static righting reflex / sensory activity / grip strength / motor activity
Sacrifice and pathology:
SACRIFICE
- Male animals: All surviving animals, following completion of the mating period (min. of 28 days treatment).
- Maternal animals: All surviving animals. Females which delivered sacrificed on lactaction days 5-7, females that failed to deliver sacrificed on post-coitum day 26

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

ORGAN WEIGHTS
The tissues indicated below were weighed (from 5 selected animals/sex/group): Cervix, prostate gland, clitoral gland, seminal vesicles, coagulation gland, testes, epididymides, uterus, ovaries, vagina, preputial gland, all gross lesions

In all remaining males the epididymides and testes were weighed.

HISTOPATHOLOGY
Histopathology was performed for 5 selected animals/sex of group 1 and 4. The tissues indicated below were prepared for microscopic examination:
Adrenal glands, Peyer's patches (jejunum, ileum) if detectable, brain (cerebellum, mid-brain, cortex), pituitary gland, caecum, preputial gland, cervix, prostate gland, clitoral gland, rectum, colon, duodenum, sciatic nerve, epididymides, seminal vesicles including coagulating gland, eyes (with optic nerve (if detectable) and Harderian gland), skeletal muscle, female mammary gland area, spinal cord - cervical/midthoracic/lumbar, femur including joint, spleen, heart, sternum with bone marrow, ileum, stomach, jejunum, testes, kidneys, thymus, thyroid including parathyroid (if detectable), liver, trachea, lung, infused with formalin, urinary bladder, lymph nodes - mandibular/mesenteric, uterus, vagina, all gross lesions

Additional histopathology:
- Testes of 5 selected males of groups 1 and 4 to examine staging of spermatogenesis
- Reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina) of all animals that failed to mate, conceive, sire or deliver healthy pups
- Stomach of 5 selected animals/sex of groups 2 and 3
- All gross lesions of all animals (all dose groups) were examined.
Other examinations:
Not relevant
Statistics:
- Dunnett-test
- Steel-test
- Fisher Exact-test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No toxicologically relevant clinical signs and no mortality occurred during the study period.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

FOOD CONSUMPTION
No toxicologically significant effects on food consumption were observed, although food consumption was slight, but significantly, decreased during day 1-8 of treatment in females (pre-mating period). The effects were however minimal and no dose-related response was observed.

HAEMATOLOGY
The significant effects observed were not considered of toxicological relevance, as they were minimal and showed no dose-related response.

CLINICAL CHEMISTRY
The significant effects observed were not considered of toxicological relevance, as they were minimal and showed no dose-related response.

NEUROBEHAVIOUR
A slight significant increase in low sensor locomotor counts was noted for males in the lowest dose group. A dose-related response was however absent and the variation remained within normal limits, indicating that the effect is not of toxicological relevance.

ORGAN WEIGHTS
No toxicologically relevant changes were noted in absolute or relative organ weight as compared to the control group, although a signficant decrease in absolute and relative thyroid weight was observed in the low dose males. This effect was considered minimal, a dose-related response was absent and no related pathology was observed.

GROSS PATHOLOGY
No significant or toxicologically relevant effects were observed at gross necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological changes were noted in the stomach of test article treated animals, but these were expected to be caused by the high pH of the dosing formulations and not considered of toxicologically relevance.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant effects observed

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

RESULTS OF TEST DOSING GROUPS
Control Low (100 mg/kg bw/day) Medium (300 mg/kg bw/day) High (1000 mg/kg bw/day)
ANALYSES
Actual concentration Not determind due to complexity of the substance
Stability Stable up until temperatures of >500 °C
Homogeneity Visually acceptable
pH of formulations 6.44 11.03 11.25 11.33
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL
PARENTAL DATA (P)
Number of animals (M/F) 10/10 10/10 10/10 10/10
Mortality (numbers) No mortality observed No mortality observed No mortality observed No mortality observed
Body weight x x x x
Food consumption x *F: Slight decrease in absolute and relative food consumption during pre-mating phase (day 1-8) *F: Slight decrease in absolute food consumption during pre-mating phase (day 1-8) x
Clinical signs F: Effects on skin (alopecia; n=3, scales; n=3 and scabs; n=3) F: Effects on skin (alopecia; n=3) F: Effects on skin (alopecia; n=3) x
Functional observations (hearing, pupils, static reflex, grip, motor activity) x *Increase in low sensor locomotor count (week 4) x x
Haematology x *F: Increase in platelet count *M: Decrease in prothrombin time x
Clinical biochemistry x *F: Decrease in cholesterol and calcium levels, *M: Decrease in albumin levels x *M: Decrease in total protein and albumin levels
Organ weights x *M: Decrease in absolute and relative thyroid weight x x
Macroscopy F: Alopecia of skin (n=1) F: Alopecia of skin (n=1), M: Foci in lungs (n=1) F: Alopecia of skin (n=1), M: Foci in lungs (n=1), foci in stomach (n=3), discolouration of ileum (n=1), discolouration of adrenal gland (n=1) and enlarged mandibular lymph node (n=1) F: Discolouration of clitoral gland (n=1), M: Foci in thymus (n=2)
Microscopy STOMACH: M: Minimal glandular inflammation (n=1) and minimal squamous epithelial hyperplasia (n=1) STOMACH: F: Minimal squamous epithelial hyperplasia (n=1), M: Minimal (n=2) and slight (n=1) glandular inflammation, minimal vacuolisation (n=2) and minimal squamous epithelial hyperplasia (n=2) STOMACH: F: Minimal squamous epithelial hyperplasia (n=1), M: Minimal (n=4) glandular inflammation, minimal vacuolisation (n=2) and minimal squamous epithelial hyperplasia (n=2) STOMACH: F: Minimal squamous epithelial hyperplasia (n=3), M: Minimal glandular inflammation (n=5), minimal vacuolisation (n=2), minimal (n=2) and slight (n=1) squamous epithelial hyperplasia
x = no effects (as compared to control group)
* = significant effect

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, no toxicologically significant effects were observed in rats treated with phosphorous slag up to a dose of 1000 mg/kg bw/day for a minium of 28 days. A NOAEL of 1000 mg/kg bw/day was therefore established.
Executive summary:

An OECD 422 test was performed to study the effect of 28-day exposure to phosphorous slag in rats.

No treatment related clinical signs or mortality were noted. All other effects noted at different examinations were minimal and not considered of toxicological significance. Histopathological changes in the stomach of test article treated parental animals are expected to be caused by the high pH of the dosing formulations and not considered toxicologically relevant.

Under the conditions of this study, no toxicologically significant effects were observed in rats exposed to a dose of up to 1000 mg/kg bw/day for a minium of 28 days. A NOAEL of 1000 mg/kg bw/day was therefore established.