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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
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: 210-218 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 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
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation:
- Proof of pregnancy: vaginal plug, sperm in vaginal smear or staging of the estrous cycle referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing mating was terminated
- After successful mating each pregnant female was caged: Individually
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
Details on study schedule:
- Age at mating of the mated animals in the study: approx. 13 weeks (P)
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 required

Examinations

Parental animals: Observations and examinations:
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

REPRODUCTIVE PERFORMANCE:
- Precoital interval
- Reproductive indices
- Length of gestation
- Number of corpora lutea and implantations
-
Oestrous cyclicity (parental animals):
Not included in this OECD test
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
Not performed

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was determined for pups born or found dead
Postmortem examinations (parental animals):
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.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and were sacrificed at approx. 4 days of age (on day 5, 6 or 7).
- These animals were subjected to postmortem macroscopic examination

GROSS NECROPSY
- Gross necropsy consisted of external examinations for abnormalities. The stomach was examined for the presence of milk. Defects or cause of death were evaluated if possible.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed (but tissues were preserved for further examination when necessary)
Statistics:
- Dunnett-test
- Steel-test
- Fisher Exact-test
Reproductive indices:
- Mating index: No. of females mated / No. of females paired * 100
- Fertility index: No. of pregnant females / No. of females paired * 100
- Conception index: No. of pregnant females / No. of females mated * 100
- Gestation index: No. of females bearing liver pups / No. pregnant females * 100
Offspring viability indices:
- % live males at first litter check: No. of live male pups at first litter check / No. of live pups at first litter check * 100
- % live females at first litter check: No. of live female pups at first litter check / No. of live pups at first litter check * 100
- % of postnatal loss day 0-4 of lactation: No. of dead pups on day 4 of lactation / No. of live pups at first litter check * 100
- Viability index: No. of live pups on day 4 of lactation / No. of pups born alive * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No toxicologically relevant clinical signs and no mortality occurred during the study period.

FOOD CONSUMPTION (PARENTAL ANIMALS)
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.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No effects observed on testes and/or epididymides weight

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The mating, fertility and conception indices, precoital time, gestation length, number of corpora lutea and implantation sites were unaffected by treatment

ORGAN WEIGHTS (PARENTAL ANIMALS)
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 and a dose-related response was absent.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No toxicologically relevant or significant findings were noted in the macroscopic examination as compared to controls

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopic changes were observed (but not significant) in the stomach of animals in each of the treatment groups, consisting of glandular inflammation, limiting ridge vacuolisation and limiting ridge squamous epithelial hyperplasia. These findings were only significant for males of the high dose group (with a positive trend). A slight dose-related trend could be identified over the treatment groups. The effects are expected to be caused by the high pH of the dosing formulations and therefore not considered of toxicological relevance.

No microscopic findings indicated infertility in the animals suspected thereof.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental
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
Dose descriptor:
NOAEL
Remarks:
reproduction
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

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
One pup of the control group and low dose group were found dead at the first litter check and missing on day 2 of lactation, but these deaths were not considered toxicologically relevant (no dose-related trend).

CLINICAL SIGNS (OFFSPRING)
No clinical signs were noted that were not within the range considered normal for pups of this age.

GROSS PATHOLOGY (OFFSPRING)
Absence of milk in the stomach was an incidental finding in the pup found dead at first litter check. The missing pup on day 2 was most likely cannabalised.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant effects observed

Overall reproductive toxicity

Reproductive 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
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
Effects on sperm (weight of testes/epididymides and staging of spermatogenesis) All stages present All stages present and no effect on organ weights All stages present and no effect on organ weights All stages present and no effect on organ weights
Precoital interval (mean duration in days) 2.4 5.1 3.0 2.8
Mating index (%) 100 100 100 100
Fertility index (%) 100 100 100 90
Conception index (%) 100 100 100 90
Number of corpora lutea (mean numbers) 17.5 14.6 15.7 14.0
No. of females with implantations (numbers) 10 10 10 9
No. of implantations (mean numbers) 13.6 12.8 12.6 13.3
Duration of gestation (mean in days) 21.2 21.2 21.4 21.1
Gestation index (%) 100 100 100 100
 
OFFSPRING TOXICITY (F1)
Number of litters 10 10 10 9
Number found dead at first litter check 1 0 0 0
Number of pups 122 117 107 113
Litter size (mean) 12.2 11.7 10.7 12.6
Sex ratio (M/F) 0.46 0.46 0.50 0.54
Mean foetal weight (M+F in gram) 6.1 6.1 6.5 6.0
Post natal survival PD0-4 (numbers) 122 116 107 113
Viability index (%) 100 99.1 100 100
Postnatal growth (BW gain PD0-4) x x x x
Macroscopic observations Dead pup: absence of milk in stomach Missing pup: most likely cannabalised x x
x = no effects (as compared to control group)
* = significant effect

Applicant's summary and conclusion

Conclusions:
Under the condtions of this study, no toxicologically significant effects were observed in P or F1 as a result of a minimum of 28-day exposure to phosphorous slag. A NOAEL of 1000 mg/kg bw/day was therefore established for developmental, reproduction and parental toxicity.
Executive summary:

An OECD 422 test was performed to study the (reproductive) 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 based. No toxicologically meaningful effects on fertility, reproductive performance or development of the offspring were observed.

Under the condtions of this study, no toxicologically significant effects were observed in P or F1 as a result of a minimum of 28-day exposure to phosphorous slag. A NOAEL of 1000 mg/kg bw/day was therefore established for developmental, reproduction and parental toxicity.