Aluminium metaphosphate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 Sep 2001 - 24 Jun 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
(1) Both substances are inorganic salts containing a trivalent aluminium cation and phosphoric acid. Thus, they all share the Al3+ cation and the PO43- anion as common functional groups.
(2) Both substances will ultimately dissociate into the common breakdown products of the Al3+ cations and the PO43- anion.
(3) In general, independently of the cation under consideration, the water solubility of phosphates decreases with increasing degree of phosphate condensation (orthophosphate > diphosphate > triphosphate > polyphosphate).
In accordance with the provisions set out in Annex XI, Section 1.5, the results of the studies used for assessment and read-across are adequate for the purpose of classification and labelling and/or risk assessment; have adequate and reliable coverage of the key parameters addressed in the corresponding test method; cover an exposure duration comparable to or longer than the corresponding test method; and adequate and reliable documentation of the applied method is provided in the technical dossier.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium dihydrogen triphosphate

EC Number:

237-714-9

EC Name:

Aluminium dihydrogen triphosphate

Cas Number:

13939-25-8

Molecular formula:

AlH2O10P3

IUPAC Name:

aluminum [oxido(phosphonatooxy)phosphoryl] phosphate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Triphosphoric acid, aluminiumsalt (1:1) (Trade name: K-Fresh 100P, Tayca Corp, Japan)
- Physical state: white powder
- Analytical purity: 94.7% (based on 63.4% P2O5 and 17.9% Al2O3)
- Impurities (identity and concentrations): based on XRD-Analysis, small amounts of Aluminium metaphosphate (Al(PO3)3) and Aluminium orthophosphate (AlPO4) are present (unknown concentration)
- Purity test date: February-March 2001 and re-analysis in June 2002
- Lot/batch No.: 10101
- Stability under test conditions: After completion of the animal study, the test substance was analysed by the manufacturer and its stability was confirmed.
- Storage condition of test material: the test material was stored in an airtight container in a cool place (actual range 2-6°C)

Test animals

Species:

rat

Strain:

other: Crj:CD(SD)IGS

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Centre of Charles River Japan
- Age at study initiation: 8 weeks on arrival, 10 weeks at study initiation
- Weight at study initiation: 359-409 g (males), 210-252 g (females), after acclimation period
- Housing: Animals were housed in metal cages (260W x 380D x 180H, mm) at 2 males per cage and 2 females per cage in the quarantine and acclimation periods, 1 animal per cage after group allocation, 1 male and 1 female per cage in the copulation period, 1 dam per cage in the gestation period and 1 litter per cage in the nursing period.
- Diet: γ-Irradiated solid fed CRF-1 from Oriental Yeast Co. Ltd., ad libitum
- Water: Sapporo city water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 50±20
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% carmellose sodium in purified water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Each dose of triphosphoric acid aluminium salt was accurately weighed, finely ground in a mortar then suspended using the control substance to the prescribed concentration and dispersed using a stirrer. A mask and rubber gloves were worn at the time of preparation and procedures were carried out on a clean bench. The prepared solution was put into an airtight container protected from light, stored in a cool dark place (actual range 2-8°C) and used in administration within 1 week after returning it to room temperature. Remaining dosing solution was disposed of by incineration.

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually (1 dam per cage during gestation and 1 litter per cage during nursing)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Preparation of dosing solution and chemical analysis

(1) Preparation of dosing solution
Each dose of triphosphoric acid aluminium salt was accurately weighed, finely ground in a mortar then suspended using the control substance to the prescribed concentration and dispersed using a stirrer. A mask and rubber gloves were worn at the time of preparation and procedures were carried out on a clean bench. The prepared solution was put into an airtight container protected from light, stored in a cool dark place (actual range 2-8°C) and used in administration within 1 week after returning it to room temperature. Remaining dosing solution was disposed of by incineration.

(2) Chemical analysis of dosing solution
The uniformity of 1 and 200mg/mL dosing solutions of triphosphoric acid aluminium salt prior to administration and the stability of 7-day storage in a cool dark place were consequently analysed using concentration analysis method as below. As a result, the uniformity of prepared solutions of 1 and 200mg/mL triphosphoric acid aluminium salt and the stability of storage for 7 days in a cool dark place were confirmed.
The results of analysis of the concentration of triphosphoric acid aluminium salt in prepared solutions at each concentration at the initial and final times of preparation and used for dosing showed a content of 100.0-105.7% which was within the judgement criterion (85-115%).

(3) Concentration analysis method
Phosphorus standard solution (P: 100mg/L, for use in water quality tests, Wako Pure Chemical Industries) was dissolved in distilled water (Wako Pure Chemical Industries), a standard solution was prepared by adding a colouring agent and absorbance (wavelength 405nm) was measured using an ultraviolet-visible spectrophotometer (UV-160A, Shimadzu Corp.).
A fixed volume of the prepared test substance solution is accurately collected, 35% sodium hydroxide solution, distilled water and nitric acid (1/2) solution were added and this formed the decomposition solution.
After centrifugation of this decomposition solution at 3500rpm, the supernatant was collected then distilled water, nitric acid (1+1) solution and a colouring agent were added and absorbance was measured.
In addition, nitric acid (1+1) solution was added to distilled water to form a blank test solution and the absorbance of this blank test solution was measured. In stability and concentration confirmation tests, the middle layer of the prepared test substance solution was sampled 3 times and the absorbance was measured once after each preparation. In the uniformity test, the prepared test substance solution upper layer and lower layer were each sampled 3 times, and the absorbance of each was measured once after each preparation.
The concentrations of AlH2P3O10x2H20 in the sample solution and in the prepared test substance solution were determined from the absorbance of the standard solution and test sample solution and the content and coefficient of variation were calculated. The content was 85-115% and the coefficient of variation was within 5% and was accepted.
Concentration of AlH2P3O10x2H20 in sample solution (mg/mL) = (absorbance of sample solution - BL)/(mean absorbance of a standard solution - BL) x
standard solution concentration (0.01mg/mL) x conversion factor

BL: mean absorbance of blank test solution
conversion factor (P → AlH2P3O10x2H20): 317.95/ (30.97x3) = 3.422

Concentration of prepared test substance solution (mg/mL) = concentration of AlH2P3O10x2H20 in sample solution x dilution factor
Dilution factor: Total volume of sample solution (mL) x total volume of decomposition solution (mL) / amount of decomposition solution collected (mL) / amount of prepared test substance solution collected (mL)

Content (%) = (mean concentration of prepared test substance solution) / (displayed concentration of prepared test substance solution) x 100
Coefficient of variation (%) = standard deviation of concentration of prepared test substance solution) / (mean concentration of prepared test substance solution) x 100

Duration of treatment / exposure:

males: 46 days (from 14 days prior to mating)
females: from 14 days prior to mating, through mating, gestation and delivery up to Day 4 of lactation

Frequency of treatment:

daily

Details on study schedule:

not appropriate for screening study

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: From the results of a preliminary 14-day experiment in which oral administration at a volume of 10mL/kg of suspensions of triphosphoric acid aluminium salt in 0.5% CMC at doses of 0, 100, 300 and 1000mg/kg were administered to 3 male and 3 female SD rats (Crj:CD(SD)IGS) per group, effects of test substance administration were not observed in any treatment group. Therefore, in both males and females, 1000mg/kg was established as the high dose, 300 and 100mg/kg were established at the intermediate and low doses in the same way as in the preliminary experiment and a group given 0.5% CMC was included as a control making a total of 4 groups.
- Rationale for animal assignment (if not random): After the completion of quarantine and acclimation, 40 healthy males and 40 healthy females (showing no abnormalities in the oestrus cycle) were selected and were used in experiments at 10 weeks of age. Group allocation was carried out using a stratified random sampling method based on body weight at the end of the quarantine and acclimation periods (the day before dosing) so that the mean body weight was uniform. The body weight range at the time of group allocation was 359-409g for males and 210-252g for females and these was within ±20% of the mean body weights (384.1g for males, 228.1g for females). Animals excluded from selection were excluded from the study.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least 4 times a day from Day 1 of administration until the autopsy on the day following Day 46 of administration.
- Cage side observations included: viability, external appearance and behaviour

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 1, 2, 5, 7, 10 and 14 of administration, every 7 days thereafter before administration, at the end of dosing and on the day of autopsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Days 43-44 of administration

For further information on parental examinations, please refer to 7.5.1 "Repeated dose toxicity"

Oestrous cyclicity (parental animals):

For evaluation of estrous cycle, vaginal smears were prepared in all females daily from day 10 before the start of administration until copulation. Determination of the stage of the estrous cycle (pro-oestrus, the period before estrus, metestrus and diestrus) was determined using a light microscope. Each stage of estrous which was repeated twice or more in the interval from day 4 to day 6 was judged to be normal and the normal estrous period was calculated. When some stages were not observed and when the stage of the same estrous cycle continues for in excess of 3 days, then an anomaly, estrous or diestrous continuing for 7 days or more were taken as constant estrous or constant diestrous which was judged to be abnormal.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
The following reproductive tissues and organs were weight and evaluated at autopsy:
testes (left and right), epididymides (left and right), prostate gland, seminal vesicles and coagulating gland (left and right)

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring: number of surviving offspring and number of dead neonates (determined once a day for each dam on day 0 to day 4), sex of pups and external appearance, weight gain (on day 0, 1 and 4), mean body weight (determined for each litter separately for males and females)


GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed on day 46 of administration.
- Maternal animals: All surviving animals were sacrificed on day 46 of administration.

GROSS NECROPSY
- Gross necropsy consisted of macroscopic evaluation of the following organs and tissues: brain (cerebrum/cerebellum), pituitary gland, thymus, thyroid (including the parathyroid gland, left and right), adrenal glands (left and right), spleen, heart, thoracic aorta, tongue, oesophagus, stomach (forestomach and glandular stomach), liver, pancreas, duodenum, jejunum, ileum (including Peyer's patches), caecum, colon, rectum, larynx, trachea, lung (including bronchus), kidneys (left and right), urinary bladder, testes (left and right), epididymides (left and right), prostate gland, seminal vesicles and coagulating gland (left and right), ovaries (left and right), uterus (horn and neck), vagina, eyeballs and Harder's glands (left and right), skin (taken from the right abdomen), sternal and femoral bone (including bone marrow), spinal cord (neck), mesenteric lymph nodes, mandibular lymph nodes (left and right), submaxillary salivary glands (left and right), sublingual glands (left and right), parotid glands (left and right), skeletal muscle (gastrocnemius muscle), sciatic nerve (right).
Additionally (males): abdominal muscle seen in an umbilical hernia (No. 404).
Additionally (females): sites of macroscopic abnormalities (including normal tissue and boundary area) as well as the spleen adhesion tissue and pancreaticosplenic lymph nodes tissue in No. 351 and left axilla skin in No. 352.

HISTOPATHOLOGY / ORGAN WEIGHTS
Males
Slices from all animal were prepared after embedding in paraffin and staining with haematoxylin-eosin then specimens from all animals in groups 1 and 4 were microscopically examined. All cases where the testes and epididymides showed changes thought to be effects of test substance administration and animals in other treatment groups were microscopically examined. The liver of 1 animal (No. 208) in the 100mg/kg group which showed abnormal finding at autopsy was also microscopically examined.
Females
The same method as used in males was carried out. A microscopic examination was carried out on the pancreas, liver, spleen, pancreaticosplenic lymph nodes and the retroperitoneum in 1 animal (No. 351) in the 300mg/kg treatment group which did not exhibit pregnancy up to day 25 of pregnancy, on the glandular stomach of 1 animal (No. 359) among all dead offspring in the 300mg/kg treatment group, and on the brain of 1 animal (No. 256) showing abnormalities in the 100mg/kg treatment group at autopsy and on the spleen and mammary gland of 1 animal (No. 352) in the 300mg/kg treatment group.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at 4 days of age following observation of the bodily appearance (including the oral cavity) .

GROSS NECROPSY
- Gross necropsy of sacrificed offspring consisted of systemic organs and tissues.
Dead animals were autopsied immediately on discovery.

Statistics:

Mean values and standard deviations were calculated. For statistical evaluation, Bartlett’s test was used for body weight/weight gain, growth rate, feed consumption, urinalysis, haematology (excluding leucocyte percentage), biochemistry, absolute and relative organ weights, estrous cycle, number of corpora lutea, number of implantation sites and implantation index, number of offspring, number of surviving offspring and dead neonates, delivery index, live birth index, sex ratio, gestation period, number of surviving offspring and viability index.
In the case of equal variance, analysis was carried out using One-Way analysis of variance and, in the case of unequal variance, the Kruskal-Wallis test.
If the results of One-Way analysis of variance showed no significant difference, a Dunnett’s test was used for further analysis. If the results of the Kruskal-Wallis method showed no significant difference, a Mann-Whitney U-test was applied.
The live birth index, sex ratio, viability index and body weights were dealt with taking 1 litter as a sample unit.
For the rate of occurrence of abnormalities in the estrous cycle, the copulation index, fertility index, gestation index, nursing index and histopathological findings showing a grade of 1 or higher, the multiple sample χ2 test was carried out followed by a 2-sample χ2 test. However, if there were mismatches in the 2-sample χ2 test, Fisher's exact test was used.
The level of significance was taken as 5% in comparison test with the control group.

Reproductive indices:

The following reproductive indices were determined:
copulation index (%): (number of paired males and females which copulated/number of males and females housed together) x 100
gestation index: (number of females with live births/number of pregnant females) x 100
fertility index (%): number of females which conceived/number of males and females which copulated) x 100
delivery index: (number of offspring/number of implantation sites) x 100
The following reproductive parameters were determined:
number of corpora lutea
number of implantation sites
gestation period (calculated as the number of days from day 0 of pregnancy (the day copulation was established) to day 0 of nursing (the day delivery was completed)
gestation index (%): number of females with live births/number of pregnant females) x 100
delivery index (%): number of offspring/number of implantation sites) x 100
implantation index (%): number of implantation sites/number of corpora lutea) x 100

Offspring viability indices:

The following offspring indices were determined:
viability index (%): (number of surviving offspring on day 4 of nursing/the number of live offspring at birth) x 100; cannibalism or unexplained disappearance of neonates was treated as a death
live birth index (%): (number of live offspring at birth/number of offspring) x 100
nursing index (%): (number of females with nursing pups on day 4 of nursing/the number of females with live births) x 100
sex ratio: number of male offspring/(the number of male offspring + the number of female offspring)

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see Details on results

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

see Details on results

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

see Details on results

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Males
Although changes were not observed in the 100 and 300mg/kg treatment groups, central abdominal swelling and soiling of the perioral fur were observed in 1 animal (No. 404) in the 1000mg/kg treatment group on the day after the final dose (day of autopsy).
Females
Although changes were not observed in the 100 and 1000mg/kg treatment groups, 1 animal in the 300mg/kg treatment group showed a subcutaneous mass on day 13 of pregnancy and thereafter.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males
Significant differences in changes in body weight, amount of body weight gain and rate of body weight gain were not seen in any group during the study period compared to the control group.
Females
Significant differences compared to the control group were not seen in body weight change, amount of body weight gain and rate of body weight gain in each treatment group in the administration period before pregnancy, gestation period and nursing period.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Not applicable (the test substance was aministered via gavage).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No effects on estrous cycle were noted in females of any dose group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Copulation index, gestation index and nursing index were comparable among the groups. In the 300mg/kg treatment group, 1 animal did not give birth up to day 25 of pregnancy. 2 dead foetuses and 5 surviving foetuses were found within the uterus of this dam at autopsy on day 26 of pregnancy. As surviving fetuses were present in the uterus and no comparable effect was observed in the higher dose group, this effect is not considered as adverse but rather incidental. Further, 1 animal delivered 7 dead foetuses on day 25 of pregnancy thereby reducing the gestation index to 90% compared to 100% in the control and remaining treatment groups, including the high-dose group. Due to the incidental occurrence in the mid-dose group, the decrease in gestation index is not considered as adverse.
Fertility index in the control, low- and mid-dose group reached 100% although 2 control and 4 mid-dose males revealed testis atrophy in histopathology. A decrease in fertility index was determined in the high-dose group, which might be related to infertility of 1 male (severe testis atrophy) and vaginal closure/inflammation in uterus horn in 1 female. Thus, due to the low incidence of infertility (1/10 males and 1/10 females), spontaneous occurrence rather than a treatment-related effect on fertility is considered as reason for infertility. Moreover, based on a comparison with historical control data (Ema et al., 2014; please refer to Table 4), the decreased fertility index observed in the high-dose group is not considered to be of biological significance as pregnancy rates ranging from 80 - 100% were evaluated for the respective tester strain and study period (depending on the feed source).
Number of corpora lutea, number of implantations, implantation index, delivery index and total number of offspring were not affected by treatment. The mid-dose group revealed slightly reduced results in regard to several parameters (incl. number of corpora lutea, number of implantations, number of dams with live offspring, total number of offspring, delivery index, surviving number and delivery index). However, due to a missing dose-response, the observed alterations are considered as incidental and not-treatment related, especially as most parameters fall into the range of historical control data (Ema et al., 2014, please refer to Table 4).

ORGAN WEIGHTS (PARENTAL ANIMALS)
Males
Significant differences were not observed in any organ in any treatment group compared to the control group.
Females
Although the absolute and relative weights of the spleen showed a significantly lower value in the 100 and 1000mg/kg treatment groups compared to the control group, significant differences were not seen in the 300mg/kg treatment group. Thus, due to a missing correlation to a dose-response, the alterations in spleen weights are not considered as adverse.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Males
Deformity of the liver and multifocal fine yellowish-white spots were seen in 1 animal (no. 208) in the 100mg/kg group.
1 Animal (No. 303) in the 300mg/kg group showed atrophy of the bilateral testes and epididymides.
In the 1000mg/kg treatment group, 1 animal (No. 401) showed atrophy of the bilateral testes and epididymides and 1 animal (No. 404) showed umbilical hernia and a dark red protrusion in part of the ileum.
Pregnancy was established in a female (No. 353) paired with No. 303 who showed atrophy of the testes and epididymides and pregnancy in a female (No. 451) paired with No. 401 was not established.
Females
In the 100mg/kg treatment group, 1 animal (No. 256) showed dilation of both cerebral ventricles.
In the 300mg/kg treatment group, 1 animal (No. 352) showed swelling of the spleen and a subcutaneous white mass. Dark reddish macules were seen in the glandular stomach in 1 animal (No. 359) which was among 7 dead offspring delivered, in 1 animal (No. 351) in which delivery was not seen on day 25 of pregnancy, swelling of the liver, deformity of the spleen, adhesion of the intraperitoneal organs (spleen, pancreas, panniculus and retroperitoneum) and swelling of the pancreaticosplenic lymph nodes were observed.
In the 1000mg/kg treatment group, 1 animal (No. 455) showed an ileal diverticulum. In one infertile animal (No. 458), closure of the vagina and dilation of the uterus were seen and retention of a yellowish-white fluid was seen within the uterus.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Males
1 Animal (No. 401) in the 1000mg/kg treatment group showed severe atrophy and interstitial oedema in the seminiferous tubules in the testes, a severe decrease in sperm in the epididymides and intermediate intraluminal cell debris, and this animal did not establish pregnancy with a paired female. Moreover, atrophy of testes of milder severity was observed in 2 control and 4 mid-dose males. However, as all affected control- and mid-dose males were fertile, including the mid-dose male with intermediate atrophy of the seminiferous tubules in the testes and an intermediate decrease in sperm in the epididymides, atrophy of testes observed in the control and mid-dose group is not considered as adverse. Due to missing dose-response and occurence in the control group, atrophy of testes observed in the high-dose group is considered rather incidental than treatment-related.
Mild myocardial degeneration was seen in 1 other animal (No. 408) in which pregnancy with a paired female was not established.
In the low dose group, no histopathological alterations were observed.
Females
In the 1000mg/kg treatment group, 1 animal (No. 256) showed dilation of the cerebral ventricles.
In the 300mg/kg group, 1 animal (No. 352) showed mild extramedullary haemopoiesis in the spleen and adenoma in the mammary gland. In 1 animal (No. 351) in which delivery was not seen, mild extramedullary haemopoiesis in the liver and spleen and intermediate necrosis in the spleen were seen, and 1 animal (No. 359) among all nursing infant deaths showed mild ulceration of the glandular stomach.
Localised necrosis of the liver and atrophy of the thymus were sporadic in the 1000mg/kg treatment group. In 2 animals (Nos. 451 and 458) for which pregnancy was not established, No. 451 showed mild vitreous casts in the kidneys and No. 458 showed mild inflammation of the uterine horn and neck of the uterus and vaginal closure. As inflammatory changes were not seen for the vaginal closure, this was judged to be congenital.

For details, please refer to Tables 1 -4 in the attachment.

Effect levels (P0)

Dose descriptor:

NOEL

Remarks:

fertility

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed on fertility (for details, please refer to "Details on results")

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:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
The number of surviving offspring on day 0 of nursing, live birth index, number of surviving offspring on day 4 of nursing and viability index were comparable among the groups. Neonatal deaths at the end of delivery were observed in the control group (2 males and 1 female) and in the 300 mg/kg treatment group (3 males, 4 females and 1 cannibalised neonate of unknown sex).
Deaths or unclear cases in the period up to day 4 of nursing was observed in the control group (4 males and 2 females), in the 100mg/kg treatment group (1 male) and in the 1000mg/kg treatment group (1 female) .

CLINICAL SIGNS (OFFSPRING)
The general condition of offspring was not affected by treatment.

BODY WEIGHT (OFFSPRING)
No effect on body weight was noted in any dose group.

PATHOLOGY (OFFSPRING)
No effects were noted in the autopsy of neonates despite, injury to the lower jaw and a skin crust observed in 1 female in the 100mg/kg treatment group and loss of the tail was seen in 1 female in the 1000mg/kg treatment group.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects noted

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

A summary of experimental results is attached (Tables 1- 3). For further details on results, please refer to 7.5.1 "Repeated dose toxicity".

Historical control data are shown in Table 4 (please refer to the attachment).

Applicant's summary and conclusion

Conclusions:

Effects of test substance administration were not seen on the reproductive ability of parents and birth of neonates in any treatment group. Consequently, the no observed effect level (NOEL) of repeated dose triphosphoric acid aluminium salt in reproduction in parents and the no observed effect level (NOEL) in the birth of neonates was considered to be 1000mg/kg/day under these experimental conditions.