Dodecaaluminium trimolybdenum dodecaoxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There are no data available on toxicity to reproduction in mammalian cells for aluminium molybdenum oxide. However, there are reliable data for aluminium and molybdenum compounds considered suitable for read-across using the analogue approach. For identifying hazardous properties of aluminium molybdenum oxide concerning human health effects, the existing forms of the target substance at very acidic and physiological pH conditions are relevant for the risk assessment. As aluminium molybdenum oxide is an inorganic metallic compound, the tendency to hydrolyze is based on its solubility which is highly pH-dependent. At the physiological pH of 7.4, the availability of aluminium is decreased due to the formation of insoluble Al(OH)₃; molybdenum species exist as molybdate anion (MoO₄^2-). At acidic pH conditions (pH < 4), aluminium is predominantly present as Al³⁺, whereas molybdenum species are primarily available in the acidic forms HMoO₄^-or H₂MoO₄. Since the release of aluminium and molybdate species is affected by the biological and pH conditions, the use of data on soluble aluminium and molybdenum compounds is justified for toxicological endpoints representing a worst case scenario. For further details, please refer to the analogue justification attached in section 13 of the technical dossier.

Molybdenum compounds

There are no data available on effects on fertility for molybdenum compounds.

However, no adverse effects on reproductive organs and tissues were observed in subchronic and chronic toxicity studies when molybdenum trioxide was given by inhalation to rats and mice of both sexes. In addition, no changes in sperm counts and motility were observed in the subchronic inhalation study in mice and rats.

 

Aluminium compounds

Test guidelines for assessing reproductive endpoints include the Reproductive/Developmental Toxicity Screening study (OECD 421), the combined Repeated Dose Toxicity Study and Reproductive/Developmental Toxicity Screening study (OECD 422), the One–Generation Reproductive Toxicity Study (OECD 415) and the Two–Generation Reproductive Toxicity Study (OECD 416). Of these, only the Two–Generation Reproductive Toxicity Study (OECD 416) provides adequate and complete information (ECHA, 2008, Chapter 7a) to meet the information requirements.

There are 2 two-generation studies available to support a hazard assessment of the developmental effects of aluminium. However, interpretation of both studies for risk assessment is limited since effects reported may be due to limited water consumption seen in the study. Therefore, other OECD Test Guidelines can be used in combination to fulfill the information requirements. Currently, there are two GLP studies on reproductive/developmental toxicity of aluminium compounds are available which are describe further down.

In the 2 OECD 416 and GLP compliant studies, aluminium sulfate Al2(SO4)3 (AS) and aluminium ammonium sulfate (AAS) (CAS#: 7784-25-0 (anhydrous)) CAS#: 7784-26-1 (dodecahydrate)] were administered by a relevant oral route with drinking water to Crl:CD(SD) rats at multiple dose levels (120, 600 and 3000 ppm and at 0, 50, 500 or 5000 ppm, respectively) before mating, during mating, gestation and lactation period in the two generation reproductive toxicity study (Hirata-Koizumi et al., 2011). Twenty-four animals per sex and group (F0 and F1 generation) were given AS and AAS in pH 3.57 - 4.20 drinking water beginning at 5 weeks of age for 10 weeks until mating, during mating, throughout gestation and lactation. Litters were normalized on PND 4. In the F1 generation, 24 male and 24 female weanlings were identified as parents on PNDs 21 to 25, ensuring an equal distribution of body weights across groups. Drinking water provided to the F1 offspring contained the identical AS/AAS concentrations as those of their parents. These animals were then mated, and followed through gestation and lactation until sacrifice on PND 26. Each female was mated with a single male receiving the same AS/AAS drinking water concentration; if successful mating did not occur (as evidenced by sperm in a vaginal smear or presence of a vaginal plug) within the two week mating period, then the female was put in with another male from the same group who had mated successfully.

Observations assessed in the parental animals included clinical signs of toxicity, estrous cycle, copulation, fertility, gestation (including numbers of implantations) and delivery indices, the numbers of testis and cauda epididymal sperm, sperm swimming speed, percentage of motile sperm, percentages of motile sperm and percentages of morphologically abnormal sperm. Litter parameters recorded at parturition (post-natal day zero; PND0) included the number of live and dead offspring and the numbers and types of gross malformations. Developmental landmarks assessed in the F1 and F2 pups were: body weight (daily); sex ratios, pinna unfolding PND1 to PND4; anogenital distance on PND 4; incisor eruption (in one male and one female pup per dam) beginning on PND 8; eye opening beginning on PND 12; surface righting reflex (PND 5), negative geotaxis (PND 8); and mid-air righting reflex (PND 18) in one male and one female pup per litter. In the F1 pups selected as F1 parents, the males were observed for timing of preputial separation (starting on PND 35) and the females were observed for timing of vaginal opening (starting on PND 25). Neurobehavioral testing was conducted at two time points in randomly selected offspring (locomotor activity and T maze test).

The major findings in the aluminium sulfate study (Hirata-Koizumi et al., 2011) include decreased drinking water consumption for both sexes in all AS groups, variable reductions in food consumption, reduced body weight in pre-weaning animals at 3000 ppm, delayed sexual maturation of the female F1 offspring at 3000 ppm, and decreased absolute liver, epididymides, thymus and spleen weight in the offspring at 3000 ppm. The authors proposed a LOAEL for aluminium sulfate for parental systemic toxicity and reproductive developmental toxicity of 31.2 mg Al/kg bw/day (3000 ppm) and NOAEL at 8.06 mg Al/kg bw/day (600 ppm). However, the authors state, correctly, that because “paired-comparison data are not available to assess the effects of decreased water intake in the absence of AS exposure” there is a possibility that the decreased absolute organ weights as well as delayed vaginal opening in the F1 females is likely secondary to the reduced body weight. The reduction in bodyweight is in turn likely to be related to the reduced food and water intake and a substance specific effect cannot be deduced from this study and the authors suggested their NOAEL was conservative.

The statistically significant delay in F1 female vaginal opening (29.5 ± 2.1 in controls and 31.4 ± 1.7 days in the highest dose group) was not accompanied by adverse changes in estrous cyclicity, anogenital distance or further reproductive performance. It is likely that the observed effects are secondary to the reduced body weight development. The authors concluded it is unlikely that “Al to have a clear impact on the hormonal event”. The AS levels added to drinking water by Hirata-Koizumi et al. (2011) were 190, 946 and 4700 times greater than Al levels found naturally in drinking water (ca. 0.1 mg/L; WHO, 2003). 

The results presented on AAS (Hirata-Koizumi et al. 2011) provide no evidence that prolonged consumption of AAS has an adverse impact on copulation, fertility and reproductive success in male and female Crl:CD(SD) rats consuming up to 517 mg AAS/kg-day. In discussing their data, Hirata-Koizumi et al. (2011) concluded that “copulation, fertility or gestation indices were not affected up to the highest dose tested at which average Al intake from food and drinking water was estimated to be 36.3 - 61.1 mg Al/kg per day.” 

The authors identified a LOAEL of 5000 mg AAS/L for both parental toxicity and reproductive toxicity (based on reduced pre-weaning body weight gain in F1 male (at PND 21) and female (PND 14, 21) pups, delay in the vaginal opening in F1 female pups, potentially attributed to inhibition of growth and decreased organ weights in F1 and F2 male and female offspring). The suggested LOAEL level corresponds to 36.3 mg Al/kg bw per day. The reported NOAEL from the Hirata-Koizumi et al. (2011) study is 500 mg AAS/L which corresponds to 5.35 mg Al/kg bw per day.

Interpretation of the results of both studies is difficult due to the clear effect of AS/AAS treatment on fluid consumption. Addition of AS to drinking water at high concentrations led to reduced pH (3.57 to 4.2) and this appears to have reduced the palatability of the drinking water. At these AS/AAS levels, the F0 and F1 females also decreased their food consumption relative to the controls. As a result, due to decreased drinking water consumption and decreased food consumption of F0 and F1 dams during the later stages of lactation, the observations reported represent secondary effects due to maternal dehydration and reduced nursing that may have influenced pup weight on PND 21. Because the effects reported could be related to decreased maternal fluid consumption, the utility of this study for risk assessment is limited.

In a GLP study, Beekhuijzen (2007) evaluated the effects of aluminium chloride (basic) (CAS# 1327-41-9) on early postnatal development in rats in a test study performed in accordance with OECD 422 (Combined Repeated Dose and Reproductive/Developmental Screening Test). Aluminium chloride (basic) was administered daily by gavage to male and female Wistar rats at doses of 0, 40, 200, 1000 mg/kg/day which contribute 0, 7.2, 36 and 180 mg Al/kg bw/day, respectively. Males were exposed to aluminium for 28 days, 2 weeks prior to mating, during mating, and up to termination; females were exposed for 37 to 53 days, 2 weeks prior to mating, during mating, during pregnancy and up to at least 3 days of lactation. Clinical signs of intoxication, mortality, body weights, food and water consumption, and reproduction process were recorded in both sexes. In addition, haematological and clinical biochemistry analyses were performed on both sexes at the end of study, together with macroscopic and microscopic examinations of the brain, thoracic and abdominal tissues and organs with special attention to the reproductive organs. Gross lesions were recorded for the cervix, clitoral gland, ovaries, uterus, and vagina in all female animals and the coagulation gland, epididymides, prepupital gland, prostate gland, seminal vesicles, and testes in all male animals. Body weights and the weights of the adrenal gland, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus were recorded for 5 animals from each group and sex. For each exposed group the following reproduction parameters were calculated: mating percentage (number of females mated x100/number of females paired); fertility index (number of pregnant females x100/number of females paired ); conception rate (number of pregnant females x100/number of females mated); gestation index (number of females bearing live pups x100/number of pregnant females); duration of gestation (number of days between confirmation of mating and the beginning of parturition); percentage of live males at first litter check (number of live male pups at first litter check x100/number of live pups at first litter check); percentage of live females at first litter check (number of live female pups at first litter check x100/number of live pups at first litter check); percentage of post-natal loss days 0 to 4 post-partum (number of dead pups on day 4 postpartum x100/number of live pups at first litter check) and viability index (number of live pups on day 4 postpartum x100/number of live pups at first litter check). The individual weights of all live pups on days 1 and 4 of lactation were measured and the sex of all pups determined by measuring the ano-genital distance. For offspring, clinical signs of intoxication and behavioural abnormalities were observed daily during at least 4 days of lactation.

No effects on developmental parameters in foetuses and offspring (growth, early development and survival) exposed to aluminium chloride (basic) at doses of 0, 40, 200 and 1000 mg/kg bw/day were reported. The NOAEL for reproductive toxicity (lack of effects on early development) proposed by the authors was 1000 mg/kg bw/day.

No treatment-related effects on locomotor activity and auditory startle response were reported in weanling male and female rats at the end of the lactation period following prenatal and postnatal (lactation) exposure to Al citrate (ToxTest, TEH-113, 2010). In the same study, no Al-citrate treatment-related effects were observed in the Functional Observational Battery tests performed on male and female rats at PND 5 and 11 (during the neonatal period) and on PND 22 (as juvenile pups).

Short description of key information:
Taking into account all available data using the analogue approach, there is only limited evidence for reproductive toxicity induced by aluminium and molybdenum compounds. Considering the data available so far, aluminium molybdenum oxide is not considered to be toxic to reproduction.

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity studies with aluminium compounds did not indicate any clear effect. Developmental effects observed could be secondary to effects on the dams. As no information is available on effects on developmental toxicity with molybdenum compounds, the available data are not sufficient for classification of aluminium molybdenum oxide. Therefore, no classification was given for the endpoint “effects on developmental toxicity” due to lacking data. 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

There are no data available on effects on developmental toxicity for aluminium molybdenum oxide. However, there are reliable data for aluminium and molybdenum compounds considered suitable for read-across using the analogue approach. For identifying hazardous properties of aluminium molybdenum oxide, the existing forms of the target substance at very acidic and physiological pH conditions are relevant for the assessment of human health effects. As aluminium molybdenum oxide is a metal-organic salt, which is only slightly soluble in water at pH 4, it is probable that the target substance has also a low degree of solubility at the physiological pH of 7.4. At acidic pH conditions, however, it can be assumed that aluminium molybdenum oxide hydrolysed into aluminium and molybdate ions in the human body. Due to the fact that the toxicological effects of aluminium molybdenum oxide are mainly caused by exposure to the molybdate ion, the use of data on soluble molybdenum compounds is justified for toxicological endpoints as a worst case scenario. In addition, various aluminium compounds are used within the read-across approach. For further details, please refer to the analogue justification attached in section 13 of the technical dossier.

Molybdenum compounds

There are no data available on effects on developmental toxicity for molybdenum compounds.

There is only the information available that no apparent accumulation of Mo in animal or human tissues occurs and only very limited amounts of Mo seem to cross the placental barrier (Vyskocil & Viau, 1999).

 

Aluminium compounds:

In terms of hazard assessment of toxic effects, available data on the toxicity to reproduction/development of other aluminium compounds was taken into account by read-across following a structural analogue approach, since the pathways leading to toxic outcomes are likely to be dominated by the chemistry and biochemistry of the aluminium ion (Al³⁺).

Domingo et al. (1989) investigated the embryotoxic and teratogenic potential of Al(OH)₃administered orally to pregnant Swiss mice. Mated female mice (20 animals per group) were administered (oral, gavage) 0, 66.5, 133 or 266 mg Al(OH)₃/kg bw/day (equivalent to 23, 46, and 92 mg Al/kg bw/day) from gestation day 6 through 15. Dams were sacrificed on gestation day 18. No sign of maternal toxicity was observed in any group based on changes in maternal weight gain, food consumption and gross signs of abnormalities at post-mortem examination. The number of total implantations, the foetal sex ratio, body weights and lengths of foetuses were not significantly affected at any of the administered doses of aluminium hydroxide. The number of early resorptions/litter was increased in all Al(OH)₃treated groups (3.0 - in the 23 mg Al/kg group, 2.4 - in the 46 mg Al/kg group, and 1.3 – in the 133 mg Al/kg group versus 0.4 in the control group) and the number of live foetuses decreased in all groups (11.1 in the control group, 9.4 in the 23 mg Al/kg group, 9.2 in the 46 mg Al/kg group and 9.8 in the 92 mg Al/kg group) (n = 18-20 litters per group). Observed effects were not considered as treatment related effects as there was no dose-response relationship observed. The Al-treated foetuses did not exhibit any marked differences in external malformations, internal soft-tissue anomalities or skeletal abnormalities compared to the controls. Suggested NOAEL is 266 mg Al/kg (lack of embryo/fetal toxicity or teratogenicity). The authors suggested that the lack of developmental toxicity of Al(OH)₃was likely due to lower gastrointestinal absorption of this compound compared with other forms of aluminium. 

A similar study was conducted by Gomez et al. (1990) in rats. Aluminium hydroxide was administered by gavage (2 times, daily) to pregnant Sprague-Dawley rats at dose levels of 192 (n = 18 animals per group), 384 (n = 18 animals per group) and 768 (n = 10 animals per group) mg/kg (equivalent to 66.5, 133 and 266 mg Al/kg bw/day, respectively) from day 6 through 15 of gestation. The animals were killed on day 20 of gestation. No adverse effects were reported on animal appearance, behaviour, maternal body weight, or absolute and relative organ weight (uterine, kidney and liver). No differences were observed for haematological and biochemical parameters but detailed results for these outcomes were not provided in +the publication. Although not statistically significant, the incidence of early resorptions was higher in all Al(OH)₃-treated groups than in the control group (0.4 - in the 46 mg Al/kg group, 1.3 - in the 92 mg Al/kg group, and 0.6 – in the 266 mg Al/kg group versus 0.0 in the control group). Increased post-implantation loss (%) was observed compared to the control group (3.6 - in the 46 mg Al/kg group, 12.5 - in the 92 mg Al/kg group, and 5.0 – in the 266 mg Al/kg group versus 0.6 in the control group).Observed changes were not considered as treatment related effects because no relationship to dose was observed. Increased post-implantation loss (2.2 times compared to the control group) was observed only in the dose 92 mg Al/kg group. Statistically significant decrease in maternal food consumption was not associated with decreased maternal body weight and no dose-response relationship was found. No Al-treatment related effects were observed on critical gestational parameters such as number of litters, corpora lutea, number of total implantations, number of live foetuses, sex ratio, or foetal body weight at any dose administered. During foetal examination, no external and visceral anomalities or skeletal malformation was detected. No significant differences in placental concentrations of aluminium were observed between the different groups. Suggested NOAEL is 266 mg Al/kg bw/day (lack of embryo/fetal toxicity or teratogenicity).

The influence of citric acid on the embryonic and/or teratogenic effects of high doses of Al(OH)₃in rats was investigated by Gómez et al. (1991).Three groups of pregnant rats were administered daily doses (gavage) of Al(OH)3 (384 mg/kg bw/day, equal to 133 mg Al/kg bw/day , n = 18), aluminium citrate (1064 mg/kg bw/day, n = 15), or Al(OH)₃(384 mg/kg bw/day, equal to 133 mg Al/kg bw/day) concurrently with citric acid (62 mg/kg bw, n = 18) on gestational days 6 to 15. A control group received distilled water during the same period (n = 17). There were no treatment-related differences on critical gestational parameters such as numbers of litters, corpora lutea, number of total implantations, number of live foetuses, sex ratio, or foetal body weight in the group treated with Al(OH)₃. No external and visceral abnormalities or skeletal malformation were detected on foetal examination. Maternal and foetal body weights were significantly reduced, the number of foetuses with delayed sternabrae and occipital ossification was significantly increased (p < 0.05), the number of foetuses with absence of xiphoides was increased in the group treated with Al(OH)₃and citric acid as compared to the control group. No significant differences in the number of malformations were detected between any of the groups (authors did not provide the quantitative data). 

Colomina et al. (1992) evaluated the influence of lactate on developmental toxicity attributed to high doses of Al(OH)₃in mice. Oral (gavage) daily doses of Al(OH)₃(166 mg/kg bw, n = 11), aluminium lactate (627 mg/kg b, n = 10), or Al(OH)3 (166 mg/kg bw) with lactic acid (570 mg/ kg bw, n = 13) were administered to pregnant mice from gestational day 6 to 15.An additional group of mice received lactic acid alone (570 mg/kg bw). A control group (n = 13) received distilled water during the same period. No signs of maternal toxicity (no statistically significant changes in food consumption, maternal body and organ weight) were observed in the dams treated with Al(OH)₃. No statistically significant treatment-related differences on critical gestational parameters such as number of litters, corpora lutea, number of total implantations, number of live foetuses, sex ratio, or foetal body weight were observed in the Al(OH)₃-treated group and no external abnormalities or skeletal malformation were detected on foetal examination. However, aluminium concentrations were significantly higher in the bones of dams, and aluminium was detected in the whole foetus of the Al(OH)₃-treated animals. Concurrent administration of Al(OH)₃and lactic acid resulted in significant reductions in maternal weight compared to the control group. In the group given lactate only, aluminium was detected in whole foetuses; however, this was not statistically different from the mean level found in the control group. Aluminium lactate administration resulted in significant decreases in maternal body weight and food consumption, foetal body weight accompanied by increases in the incidence of cleft palate. Delayed ossification was also observed in the aluminium lactate-treated animals. Although not statistically significant, the incidence of skeletal variations was higher in the group concurrently administered Al(OH)₃and lactic acid than in the control group. No other signs of developmental toxicity were detected in the Al(OH)₃and lactic acid group. In the lactiv acid group no changes in maternal body weight were observed during the gestation period although food consumption was significantly decreased in early treatment (GD 6-9, P < 0.05; GD 6-15, P < 0.01) and post- treatment periods (GD15-18, P < 0.05).Additionally increased numbers of dead foetuses and litters with dead foetuses were seen but not significant. An increased number of foetuses with delayed ossification (10/4 vs 0/0 compared to control, P < 0.05) were also observed.

In a similar experiment, Colomina et al. (1994) assessed the effect of concurrent ingestion of high doses of Al(OH)₃and ascorbic acid on maternal and developmental toxicity in mice. Three groups of pregnant mice were given daily doses (gavage, 2 times daily) of Al(OH)₃(300 mg/kg bw or 103.8 mg Al/kg), ascorbic acid (85 mg/kg bw), or Al(OH)₃concurrent with ascorbic acid (85 mg/kg bw) from gestational day 6 to day 15. A fourth group of animals received distilled water and served as the control group. The animals were killed on gestation day 18. The number of litters, corpora lutea, number of total implantations, number of live foetuses, sex ratio, and foetal body weight did not differ between the control and Al(OH)₃-treated groups. No external and visceral abnormalities or skeletal malformations were detected on foetal examination. Placenta and kidney concentrations of aluminium were significantly higher in mice receiving Al(OH)₃and Al(OH)₃plus ascorbic acid than in controls. No information was provided on the number of dams and litters in the Al-treated and control groups.

In summary, available studies indicate that aluminium hydroxide did not produce neither maternal nor developmental toxicity when it was administered by gavage during the critical period of embryogenesis (GD 6-15) to mice at doses up to 92 mg Al/kg bw/day (Domingo et al., 1989) or to rats at doses up to 266 mg Al/kg bw/day (Gomez et al., 1990). The developmental toxicity of aluminium following the oral route of exposure is highly dependent on the form of aluminium and the presence of organic chelators that influence bioavailability.

For all the studies with aluminium hydroxide, dose administration was by gavage, which would be expected to result in higher blood levels than dietary administration or administration via the drinking water, and very high dosages were used (ca. 200 – 2000x normal human exposure). Part of the reason for using such high dosages was the low solubility and bioavailability of aluminium hydroxide and the limited sensitivity of available analytical methods to determine small changes from endogenous levels of aluminium. However, the achieved dose of aluminium in maternal plasma was not measured in any of the studies reviewed. In the studies with aluminium administered in the diet or drinking water, dosages were generally identified in terms of the target dose, e.g. 1000 µg Al/g diet, without calculation of the actual dose administered based on the food or water consumption. Further, for the majority of the studies, there was no assessment of the background levels of aluminium in the food and water provided for the animals.

These factors generally lead to the conclusion that the dosages used in reproductive toxicity studies to date have been much greater than those that would be encountered in the human consumer or worker situation. In addition, the actual dose administered has usually been under-estimated because background aluminium levels in the diet and drinking water provided for the animals have not been taken into account. 

None of the studies on aluminium hydroxide showed any clear evidence of dose related developmental toxicity despite using daily dose levels up to 2000 fold higher than the normal aluminium levels of intake. Since bioavailability studies have shown that the absorption of aluminium oxide is less than that of aluminium hydroxide, it is unlikely that these would show any evidence of developmental toxicity at similar dose levels (Sullivan, 2010).

A recent combined one-year developmental and chronic neurotoxicity study with Al-citrate (Poirier et al, 2011) may be of interest for the evaluation of the neurotoxicity of Aluminium hydroxide, taking into consideration the tenfold lower bioavailability of Al-hydroxide compared to Al-citrate and excluding effects that can likely be related to the salt rather than the cation. The study was conducted according to OECD TG 426 and GLP, and the exposure covered the period from gestation day 6, lactation and up to 1 year of age of the offspring. Pregnant Sprague-Dawley dams (n=20 per group) were administered aqueous solutions  via drinking water of  3225 mg/Al citrate/ kg bw/day (300 mg Al/kg bw/day); 1075 mg/Al citrate/kg bw/day (100 mg Al/kg bw/day); 322.5 mg/Al citrate/kg bw/day (30 mg Al/kg bw/day). The highest dose was a saturated solution of Al-citrate. Two control groups received either a sodium citrate solution (citrate control with 27.2 g/L, equimolar in citrate to the high dose Al-citrate group) or plain water (control group). The Al citrate and Na-citrate were administered to dams ad libitum via drinking water from gestation day 6 until weaning of offspring. Litter sizes were normalized (4 males and 4 females) at postnatal day (PND) 4. Weaned offspring were dosed at the same levels as their dams. Dams were sacrificed at PND 23. At PND 4  1 male and 1 female pup of each litter  were allocated to 4 testing groups: D23-sacrifice group for pre-weaning observations and D23 neuropathology, D64, D120 and D365 postweaning groups for post weaning observations and neuropathology at the respective days of sacrifice. Endpoints and observations in the dams included water consumption, body weight, morbidity and mortality and a Functional Observational Battery (FOB) (GD 3 and 10, PND 3 and 10). Pups were examined daily for morbidity and mortality. Additional neurobehavioral tests were performed at specified intervals and included, T-maze, Morris water maze, auditory startle, and motor activity. Female pups were monitored from PND26 for vaginal opening, male pups from day 35 for preputial separation. Clinical chemical and haematological analysis was performed for each group on the day of scheduled sacrifice. Al-concentrations were determined in blood, brain, liver, kidney, bone and spinal cord tissues by inductively coupled plasma mass spectrometric analysis. Further metals such as iron, manganese, copper and zinc were also determined. The pathological investigation includes rain weight and neuropathology. Statistical analyses were performed using the SAS software release 9.1. Data collected on dams and pups were analysed separately. All analysis on pups was performed separately for each sex. Statistical significance was declared from P ≤ 0.05.

Results: Dams: Eight high dose dams developed diarrhoea. In the Na-citrate group one dam stopped nursing and the pups were euthanized. No significant differences between mean body weights of dosed animals compared to controls were observed during gestation and lactation. During gestation and lactation low and mid dose group animals consumed considerably more fluid than controls and high dose group animals. This is not considered treatment related as there was no dose response. In all animals the target dose was exceeded during lactation due to the physiologically increased fluid consumption.

Pups: During the pre-weaning phase weights of mean body weights of male and females in the sodium citrate and high dose group were significantly lower than the untreated controls. This suggests a citrate rather than Al-related effect. No differences between treated and control animals were observed in the FOB. No other clearly treatment related effects were observed pre-weaning.

F1-postweaning: General toxicity

No significant differences in body weights throughout the study were observed between low and mid-dose animals sodium-citrate and untreated controls. High dose males had significant lower body weights than controls by PND 84. These animals also had clinical signs. At necropsy urinary tract lesions were observed in the animals of the high dose group, most pronounced in the males, hydronephrosis, uretal dilatation, obstruction and/or presence of calculi. All high dose males were sacrificed on study day 98. The effect is probably due to Al-citrate calculi precipitating in the urinary tract at this high dose level. This effect is related to the citrate salt and cannot be attributed to the Al-ion. Female high dose animals showed similar urinary tract lesions, but with a lower incidence and severity. Urinary tract lesions were also observed in single mid dose males, but also in a few sodium citrate and control animals. Fluid consumption during the study was increased in the sodium citrate and Al-citrate groups (in particular high and mid dose) compared to controls. This is probably due to the high osmolarity of the dosing solutions. However, the consumed dose levels decreased in all dose groups during the study. In the beginning the target dose was considerably exceeded, while versus the end of the study it was considerably below the target dose.  According to the authors the assigned dose levels still remain valid.

In sodium citrate controls and high dose males and females the number of days to reach preputial separation or vaginal opening was longer than in untreated control animals. This may be related to the lower body weights in these animals at the respective time-point. As the sodium citrate group showed similar retardation this effect cannot be allocated to the aluminium cation.

No consistent treatment related effects that could be related to Al-ion exposure were observed in the FOB. No treatment related effects on autonomic or sensimotoric function were observed in the study. A weak association between Al exposure and reduced home cage activity, a very weak association with excitability, some association with neuromuscular performance were reported but according to the authors this may also be related to group differences in body weights, and an association with physiological function and is thus not considered clearly treatment related. No treatment related effect on general motor behaviour was observed. No clearly treatment related effect on auditory startle response was observed. There was no evidence of any treatment related effect on learning and memory in the Morris Water Maze test and no clearly treatment related effects in the T-maze test. Hind limb grip strength and to a lesser extend foot splay were reported to be reduced compared to controls in high and mid dose male and female animals, more pronounced in younger than in older  rats. However, the observed effects can be related to the lower body weights of the individual animals undergoing this test. No details on the individual findings and historical control data are available. It can therefore not be concluded with certainty that the observed neuromuscular effects are primary effects of the treatment and attributable to Al3+. The NOAEL was reported based on this effect as 30 mgAl/kg bw in a conservative approach.

Haematology: No clinically significant differences in hematology were observed at the investigation on day 23. In day 64 and 120 females and day 64 males the high dose group showed slight reduction in hematocrit (males only), mean hemoglobin and mean corpuscular cell volume. No such changes were observed in the 364 day group.

Clinical chemistry: while a number of borderline statistically significant changes were observed, such as globuline levels, alkaline phosphatase and glucose in the high dose group little or no biological significance is associated with them. Elevated creatinine and urea levels in Day 64 males are consistent with the renal toxicity observed in these animals.

Organ weights: Brain weights did not differ among the groups, with two exceptions in the day 64 group males brain weights were significantly lower than controls. In the 120 day female high dose group brain weights were also significantly lower than controls. These findings were not reproduced at the other sacrifice times. Brains to body weight ratios were not significantly different and the lower brain weights can be attributed to the body weight.

Pathology: The main pathology findings were the renal lesions with precipitates in the urinary tract and secondary lesions such as hydronephrosis and uretal dilatation   in particular in the high dose group males and to a lesser extend females. Fluid colonic content was also observed in some high dose animals, in particular males. According to the authors the test item clearly precipitated in the urinary tract causing stone formation and blockage and resulted in fluid colonic content. No other macroscopic effects were observed in other organs.

Histopathology: No treatment related histopathological effects were observed in the nervous system at any time point.

Aluminium concentrations in different organs were dose related. Tissue concentrations were highest in blood, and then in decreasing order brainstem, femur, spinal cord, cerebellum, liver cerebral cortex.

A conservative NOAEL of  322 mg Al-citrate/kg bw  corresponding to 30 mg Al/kg bw was derived from this study (with a bioavailability correction this would correspond to ca. 300 mg Al from Al(OH)₃.

The most important effects were however related to a precipitation of the citrate in the kidneys and urinary tract and this effect is not related to the Al³⁺ion.  The effects on grip strength and foor splay observed can also not be attributed unequivocally to Al-exposure as they may have been secondary to the general toxicity and body weight differences between treated and control animals undergoing this test. Neurobehavioral effects as reported by e.g. Thorne et al., 1986 could not be confirmed in this study.

Justification for classification or non-classification

Based on the read-across within an analogue approach, the available data on effects on fertility do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.

No data are available on developmental toxicity of molybdenum compounds. Therefore, no classification was given for this endpoint due to lacking data.