Registration Dossier

In a two-generation reproduction study (OECD 416) aluminium sulfate was administered to Crl:CD(SD) rats in water at dose levels of 0, 120, 600 and 3000 ppm (0, 8.6, 41.0 and 188 mg/kg bw/day). Based on the findings, the resulting NOAEL was 600 ppm (41.0 mg/kg bw/day). In a second two-generation study continuously exposing rats to aluminium ammonium sulfate via drinking water resulted in a NOAEL level of 500 ppm, which equals a dose of 5.35 mg aluminium/kg bw/day.

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see below

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: see below

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

see below

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

see below

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
- 120 ppm group: 1 F1 male was found dead at 9 weeks of dosing. In this animal, soiling of periocular and perinasal fur and decreased locomotor activity were observed before death. At autopsy, various changes, including accumulation of ascitic and pleural fluid and dark purple discoloration of the liver and kidneys, were found.
- 600 ppm group: 1 F0 female was found dead at 2 weeks of gestation. A subcutaneous mass was observed in the abdominal region of this animal from the beginning of 5 weeks of dosing. No abnormality was found on gross interal examination.
- 3000 ppm group: 1 F1 male was found dead at 12 weeks of dosing without any clinical sign of toxicity. No abnormality was found on gross internal examination.
No significant difference was seen between control and AS-treated groups in the incidence of clinical signs of toxicity in either male or female F0 and F1 rats (No data shown).

BODY WEIGHT, WATER AND FOOD CONSUMPTION (PARENTAL ANIMALS)
F0:
In F0 males and females of all AS-treated groups, water consumption was significantly lower than in controls almost throughout the dosing period. In F0 males, there were significant decreases in food consumption in the first week of dosing at 600 and 3000 ppm, and during week 8 and weeks 13–14 of dosing at 3000 ppm. Food consumption of F0 females showed a significantly lower value during week 1 of dosing at 3000 ppm and during week 3 of lactation at 600 and 3000 ppm. The body weight of F0 males and females was significantly lowered in the first 2 or 3 weeks of dosing at 3000 ppm.

F1:
Water consumption was significantly decreased through the dosing period in 600 ppm and 3000 ppm treated males, and during weeks 3–6, week 8 and week 10 of dosing in 120 ppm treated males. In F1 females, significant reductions in water consumption were found almost throughout the dosing period at 3000 ppm, during week 10 of dosing and week 3 of lactation at 600 ppm, and during weeks 9–10 of dosing at 120 ppm. Food consumption was significantly decreased during week 10 of dosing in F1 males of the 600 and 3000 ppm groups, and during week 3 of lactation in F1 females of the same groups. There was also a transient significant increase in food consumption during week 6 of dosing in F1 females of the 120 ppm group. The body weight of F1 males and females exhibited no significant differences between the control and AS-treated groups, except that F1 females of the 120 ppm group had significantly higher body weight during weeks 6–8 of dosing.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Based on water consumption and body weight, daily AS intakes during the premating and postmating periods in males and during the premating, gestation and lactation periods in females were calculated for each of the AS-treated groups.
Calculated mean AS intakes during the whole of these period were:
8.6, 41.0 and 188 mg/kg bw/day in F0 males,
14.4, 71.5 and 316 mg/kg bw/day in F0 females,
10.7, 50.2 and 232 mg/kg bw/day in F1 males, and
15.3, 74.2 and 338 mg/kg bw/day in F1 females,
in the 120, 600 and 3000 ppm groups, respectively.

The total ingested dose of aluminium from drinking water and food combined was estimated from the water and food consumption and body weight. Average aluminium intake was
1.62, 2.96, 8.06 and 31.2 mg Al/kg bw/day in F0 males,
2.29, 4.50, 13.5 and 52.0 mg Al/kg bw/day in F0 females,
1.93, 3.55, 9.78 and 38.5 mg Al/kg bw/day in F1 males, and
2.35, 4.72, 14.0 and 55.6 mg Al/kg bw/day in F1 females
for control through high-dose groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
During the premating period, AS produced no significant deviations in the estrous cycle of F0 and F1 females although a few control and AS-treated rats had persistent diestrus. The incidence of females with a normal estrous cycle also did not change significantly in either generation (data not shown).

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Sperm parameters examined for scheduled sacrificed adults, in F0 generation, the absolute number of cauda epididymal sperm was significantly decreased at 3000 ppm (253.8±61.3×10^6/cauda versus 286.3±40.3×10^6/cauda in the control); however, no significant changes were found in the number per gram of tissue. No such change was observed in F1 adults. There were no significant differences in the number of testis sperm, the percentage of motile sperm and progressively motile sperm, the swimming speed and pattern, and the percentage of morphologically abnormal sperm between control and AS-treated groups in either F0 or F1 adults (data not shown).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
During the mating period, copulation was not observed in two males each in the control, 120 ppm and 3000 ppm groups and in one female of the control group in the F0 generation. In the F1 generation, one male in the control group, two males and one female in the 120 ppm group, one male in the
600 ppm group, and three males and one female in the 3000 ppm group did not copulate. Among females with successful copulation, one female each in the control and 3000 ppm group and two females at 120 ppm in the F0 generation and two females each in the control, 600 ppm and 3000 ppm groups, and four females at 120 ppm in the F1 generation were not impregnated. In addition, one pregnant F0 female each at 120, 600 and 3000 ppm and one pregnant F1 female at 120 ppm did not deliver live pups; however, there were no significant differences in the copulation, fertility or gestation index, and the precoital interval or gestation length between the control and AS-treated groups in F0 and F1 generation. No significant changes were observed in the number of implantations or pups delivered, and delivery index in either generation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In F0 males, absolute and relative liver weights were significantly decreased at 3000 ppm. Absolute spleen weight was also decreased significantly in this group, but no significant change was found in the relative weight.
In F1 males, the absolute weights of the adrenals at 3000 ppm and the testes at 600 ppm were significantly decreased without significant changes in the relative weight.
There were no significant changes in the absolute and relative weights of any organ in F0 and F1 female adults (data not shown).

GROSS PATHOLOGY (PARENTAL ANIMALS)
No dose-related gross lesions were found in F0 or F1 adults.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological examination of the reproductive organs revealed no compound-related alterations. There was no significant difference in the number of primordial follicles in the ovary of F1 females between control and 3000 ppm groups (data not shown).

Dose descriptor:

NOAEL

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No compound-related changes in other reproductive/developmental parameters, including developmental neuro-behavioural endpoints.

Dose descriptor:

LOAEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Body weight was transiently decreased in the 3000 ppm dose group; In F1 and F2 pups, pre-weaning body weight gain was inhibited at 3000 ppm and the liver and spleen weights were decreased at weaning.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see below

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: see below

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

see below

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

see below

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
- 120 ppm group: 1 F1 male was found dead at 9 weeks of dosing. In this animal, soiling of periocular and perinasal fur and decreased locomotor activity were observed before death. At autopsy, various changes, including accumulation of ascitic and pleural fluid and dark purple discoloration of the liver and kidneys, were found.
- 600 ppm group: 1 F0 female was found dead at 2 weeks of gestation. A subcutaneous mass was observed in the abdominal region of this animal from the beginning of 5 weeks of dosing. No abnormality was found on gross interal examination.
- 3000 ppm group: 1 F1 male was found dead at 12 weeks of dosing without any clinical sign of toxicity. No abnormality was found on gross internal examination.
No significant difference was seen between control and AS-treated groups in the incidence of clinical signs of toxicity in either male or female F0 and F1 rats (No data shown).

BODY WEIGHT, WATER AND FOOD CONSUMPTION (PARENTAL ANIMALS)
F0:
In F0 males and females of all AS-treated groups, water consumption was significantly lower than in controls almost throughout the dosing period. In F0 males, there were significant decreases in food consumption in the first week of dosing at 600 and 3000 ppm, and during week 8 and weeks 13–14 of dosing at 3000 ppm. Food consumption of F0 females showed a significantly lower value during week 1 of dosing at 3000 ppm and during week 3 of lactation at 600 and 3000 ppm. The body weight of F0 males and females was significantly lowered in the first 2 or 3 weeks of dosing at 3000 ppm.

F1:
Water consumption was significantly decreased through the dosing period in 600 ppm and 3000 ppm treated males, and during weeks 3–6, week 8 and week 10 of dosing in 120 ppm treated males. In F1 females, significant reductions in water consumption were found almost throughout the dosing period at 3000 ppm, during week 10 of dosing and week 3 of lactation at 600 ppm, and during weeks 9–10 of dosing at 120 ppm. Food consumption was significantly decreased during week 10 of dosing in F1 males of the 600 and 3000 ppm groups, and during week 3 of lactation in F1 females of the same groups. There was also a transient significant increase in food consumption during week 6 of dosing in F1 females of the 120 ppm group. The body weight of F1 males and females exhibited no significant differences between the control and AS-treated groups, except that F1 females of the 120 ppm group had significantly higher body weight during weeks 6–8 of dosing.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Based on water consumption and body weight, daily AS intakes during the premating and postmating periods in males and during the premating, gestation and lactation periods in females were calculated for each of the AS-treated groups.
Calculated mean AS intakes during the whole of these period were:
8.6, 41.0 and 188 mg/kg bw/day in F0 males,
14.4, 71.5 and 316 mg/kg bw/day in F0 females,
10.7, 50.2 and 232 mg/kg bw/day in F1 males, and
15.3, 74.2 and 338 mg/kg bw/day in F1 females,
in the 120, 600 and 3000 ppm groups, respectively.

The total ingested dose of aluminium from drinking water and food combined was estimated from the water and food consumption and body weight. Average aluminium intake was
1.62, 2.96, 8.06 and 31.2 mg Al/kg bw/day in F0 males,
2.29, 4.50, 13.5 and 52.0 mg Al/kg bw/day in F0 females,
1.93, 3.55, 9.78 and 38.5 mg Al/kg bw/day in F1 males, and
2.35, 4.72, 14.0 and 55.6 mg Al/kg bw/day in F1 females
for control through high-dose groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
During the premating period, AS produced no significant deviations in the estrous cycle of F0 and F1 females although a few control and AS-treated rats had persistent diestrus. The incidence of females with a normal estrous cycle also did not change significantly in either generation (data not shown).

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Sperm parameters examined for scheduled sacrificed adults, in F0 generation, the absolute number of cauda epididymal sperm was significantly decreased at 3000 ppm (253.8±61.3×10^6/cauda versus 286.3±40.3×10^6/cauda in the control); however, no significant changes were found in the number per gram of tissue. No such change was observed in F1 adults. There were no significant differences in the number of testis sperm, the percentage of motile sperm and progressively motile sperm, the swimming speed and pattern, and the percentage of morphologically abnormal sperm between control and AS-treated groups in either F0 or F1 adults (data not shown).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
During the mating period, copulation was not observed in two males each in the control, 120 ppm and 3000 ppm groups and in one female of the control group in the F0 generation. In the F1 generation, one male in the control group, two males and one female in the 120 ppm group, one male in the
600 ppm group, and three males and one female in the 3000 ppm group did not copulate. Among females with successful copulation, one female each in the control and 3000 ppm group and two females at 120 ppm in the F0 generation and two females each in the control, 600 ppm and 3000 ppm groups, and four females at 120 ppm in the F1 generation were not impregnated. In addition, one pregnant F0 female each at 120, 600 and 3000 ppm and one pregnant F1 female at 120 ppm did not deliver live pups; however, there were no significant differences in the copulation, fertility or gestation index, and the precoital interval or gestation length between the control and AS-treated groups in F0 and F1 generation. No significant changes were observed in the number of implantations or pups delivered, and delivery index in either generation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In F0 males, absolute and relative liver weights were significantly decreased at 3000 ppm. Absolute spleen weight was also decreased significantly in this group, but no significant change was found in the relative weight.
In F1 males, the absolute weights of the adrenals at 3000 ppm and the testes at 600 ppm were significantly decreased without significant changes in the relative weight.
There were no significant changes in the absolute and relative weights of any organ in F0 and F1 female adults (data not shown).

GROSS PATHOLOGY (PARENTAL ANIMALS)
No dose-related gross lesions were found in F0 or F1 adults.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological examination of the reproductive organs revealed no compound-related alterations. There was no significant difference in the number of primordial follicles in the ovary of F1 females between control and 3000 ppm groups (data not shown).

Dose descriptor:

NOAEL

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No compound-related changes in other reproductive/developmental parameters, including developmental neuro-behavioural endpoints.

Dose descriptor:

LOAEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Body weight was transiently decreased in the 3000 ppm dose group; In F1 and F2 pups, pre-weaning body weight gain was inhibited at 3000 ppm and the liver and spleen weights were decreased at weaning.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

see below

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
See table 2
No significant changes were found in the sex ratio of pups and the viability index in either generation.

CLINICAL SIGNS (OFFSPRING)
For the physical development of male and female F1 pups and male F2 pups, there was no significant difference in the completion rate of pinna unfolding, and the age at completion of incisor eruption and eye opening between the control and AS-treated groups. In female F2 pups, the completion rate of pinna unfolding on PND 2 was significantly lower in the 600 ppm group (17.0±35.4 %, compared with 45.8±46.9 in controls), but no dose dependency was observed in this change. No significant changes were found in the completion rate of pinna unfolding on PND 1, 3 or 4 and in other physical developmental landmarks in female F2 pups. The AGD and AGD per cube root of the body weight ratio were not significantly different between control and AS-treated groups in male and female F1 and F2 pups (data not shown).
All male and female F1 pups in all groups achieved the surface righting reflex on PND5, negative geotaxis reflex on PND8 and midair righting reflex on PND 18. No significant changes were observed in the response time of surface righting and negative geotaxis reflex. In F2 pups, one female of the 600 ppm group failed in one of three trials of the mid-air righting reflex on PND 18; however, there was no significant difference in the mean success rate between the control and 600ppm groups (100±0.0 % versus 98.4±7.3 %). The surface righting reflex on PND 5 and negative geotaxis reflex on PND 8 were achieved in all male and female F2 pups in all groups, and no significant changes were found in the response time (data not shown).

BODY WEIGHT (OFFSPRING)
See table 2
In the 3000 ppm group, the body weight of male and female F1 pups was significantly lower than the control on PND 21. Body weights of F2 female pups were also significantly lower than controls on PND 21 at 3000 ppm. There were no significant differences in the body weight of male F2 pups between the control and AS-treated groups during the preweaning period.

SEXUAL MATURATION (OFFSPRING)
As for the sexual development of F1 male and female animals, vaginal opening was significantly delayed at 3000 ppm (31.4±1.7, compared to 29.5±2.1 in control). At this dose, body weight at the time of vaginal opening was slightly heavier than the control (119.0±13.3 g versus 109.6±11.6 g) although not statistically significant.
No significant differences between control and AS-treated groups were noted in the age at preputial separation or body weight at the time of completion in males.

ORGAN WEIGHTS (OFFSPRING)
F1: See table 3
The 3000 ppm treated males and females had a significantly lower body weight at scheduled sacrifice than the controls. In this group, absolute and relative liver weights were significantly lower than the controls. Absolute spleen weight was also decreased significantly in both sexes of the 3000 ppm group, accompanied by a significant decrease in the relative weight in males. In addition, significant decreases in the absolute weight were found for the thymus in both sexes and for the kidneys, testes and epididymides in males at 3000 ppm, and for the uterus in females at 600 and 3000 ppm. Relative brain weight was significantly increased in both sexes of the 3000ppm group.
F2: See table 4
The mean body weight at scheduled sacrifice was significantly lowered in both sexes of the 3000 ppm group. In males, the absolute and relative weights of the thymus and spleen were significantly decreased in the 3000 ppm group. Significant decreases were also found in the absolute weight of the liver and epididymides at 3000 ppm. The relative brain weight was significantly increased at this dose. At 120 ppm, the only significant change was a non-dose-related decrease in the relative thymus weight. In F2 females, there were significant decreases in the absolute and relative weights of the liver, and the absolute weight of the spleen, ovary and uterus, and a significant increase in the relative brain weight at 3000 ppm. In addition, a significant decrease in the absolute brain weight was observed only in the 600 ppm group.

GROSS PATHOLOGY (OFFSPRING)
Gross examination of delivered pups revealed one F1 pup with trauma in the perianal region and tail in the control group and one F1 pup with hemimelia and oligodactyly in the 120 ppm group, but no significant difference was found in the incidence between the control and AS-treated groups. No malformed F2 pups were found in any groups.
External and internal gross observations revealed no compound-related alterations either in F1 and F2 weanlings or in pups found dead during the preweaning period.

HISTOPATHOLOGY (OFFSPRING)
There were no dose-related histopathological changes in the liver and spleen of male and female F1 and F2 weanlings.

OTHER FINDINGS (OFFSPRING) - Behavioral effects:
Spontaneous locomotor activity at 10 min intervals and for 60 min was not significantly different between control and AS treated groups in male and female F1 rats. In the water-filled T-maze test, pre-test swimming trials in the straight channel revealed that all male and female F1 rats in each group could swim satisfactorily, and no significant changes were observed in the elapsed time to traverse the straight channel. On days 2–4 of the T-maze test, no significant changes were observed in the elapsed time and number of errors in males. In females, the elapsed time and the number of errors on day 2 of the T-maze was significantly lowered at 600 ppm, but there were no significant differences in the elapsed time or number of errors on days 3 and 4 of the T-maze test between control and AS-treated groups (data not shown).

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No compound-related changes in other reproductive/developmental parameters, including developmental neuro-behavioural endpoints.

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Body weight was transiently decreased in the 3000 ppm dose group; In F1 and F2 pups, pre-weaning body weight gain was inhibited at 3000 ppm and the liver and spleen weights were decreased at weaning.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
See table 2
No significant changes were found in the sex ratio of pups and the viability index in either generation.

CLINICAL SIGNS (OFFSPRING)
For the physical development of male and female F1 pups and male F2 pups, there was no significant difference in the completion rate of pinna unfolding, and the age at completion of incisor eruption and eye opening between the control and AS-treated groups. In female F2 pups, the completion rate of pinna unfolding on PND 2 was significantly lower in the 600 ppm group (17.0±35.4 %, compared with 45.8±46.9 in controls), but no dose dependency was observed in this change. No significant changes were found in the completion rate of pinna unfolding on PND 1, 3 or 4 and in other physical developmental landmarks in female F2 pups. The AGD and AGD per cube root of the body weight ratio were not significantly different between control and AS-treated groups in male and female F1 and F2 pups (data not shown).
All male and female F1 pups in all groups achieved the surface righting reflex on PND5, negative geotaxis reflex on PND8 and midair righting reflex on PND 18. No significant changes were observed in the response time of surface righting and negative geotaxis reflex. In F2 pups, one female of the 600 ppm group failed in one of three trials of the mid-air righting reflex on PND 18; however, there was no significant difference in the mean success rate between the control and 600ppm groups (100±0.0 % versus 98.4±7.3 %). The surface righting reflex on PND 5 and negative geotaxis reflex on PND 8 were achieved in all male and female F2 pups in all groups, and no significant changes were found in the response time (data not shown).

BODY WEIGHT (OFFSPRING)
See table 2
In the 3000 ppm group, the body weight of male and female F1 pups was significantly lower than the control on PND 21. Body weights of F2 female pups were also significantly lower than controls on PND 21 at 3000 ppm. There were no significant differences in the body weight of male F2 pups between the control and AS-treated groups during the preweaning period.

SEXUAL MATURATION (OFFSPRING)
As for the sexual development of F1 male and female animals, vaginal opening was significantly delayed at 3000 ppm (31.4±1.7, compared to 29.5±2.1 in control). At this dose, body weight at the time of vaginal opening was slightly heavier than the control (119.0±13.3 g versus 109.6±11.6 g) although not statistically significant.
No significant differences between control and AS-treated groups were noted in the age at preputial separation or body weight at the time of completion in males.

ORGAN WEIGHTS (OFFSPRING)
F1: See table 3
The 3000 ppm treated males and females had a significantly lower body weight at scheduled sacrifice than the controls. In this group, absolute and relative liver weights were significantly lower than the controls. Absolute spleen weight was also decreased significantly in both sexes of the 3000 ppm group, accompanied by a significant decrease in the relative weight in males. In addition, significant decreases in the absolute weight were found for the thymus in both sexes and for the kidneys, testes and epididymides in males at 3000 ppm, and for the uterus in females at 600 and 3000 ppm. Relative brain weight was significantly increased in both sexes of the 3000ppm group.
F2: See table 4
The mean body weight at scheduled sacrifice was significantly lowered in both sexes of the 3000 ppm group. In males, the absolute and relative weights of the thymus and spleen were significantly decreased in the 3000 ppm group. Significant decreases were also found in the absolute weight of the liver and epididymides at 3000 ppm. The relative brain weight was significantly increased at this dose. At 120 ppm, the only significant change was a non-dose-related decrease in the relative thymus weight. In F2 females, there were significant decreases in the absolute and relative weights of the liver, and the absolute weight of the spleen, ovary and uterus, and a significant increase in the relative brain weight at 3000 ppm. In addition, a significant decrease in the absolute brain weight was observed only in the 600 ppm group.

GROSS PATHOLOGY (OFFSPRING)
Gross examination of delivered pups revealed one F1 pup with trauma in the perianal region and tail in the control group and one F1 pup with hemimelia and oligodactyly in the 120 ppm group, but no significant difference was found in the incidence between the control and AS-treated groups. No malformed F2 pups were found in any groups.
External and internal gross observations revealed no compound-related alterations either in F1 and F2 weanlings or in pups found dead during the preweaning period.

HISTOPATHOLOGY (OFFSPRING)
There were no dose-related histopathological changes in the liver and spleen of male and female F1 and F2 weanlings.

OTHER FINDINGS (OFFSPRING) - Behavioral effects:
Spontaneous locomotor activity at 10 min intervals and for 60 min was not significantly different between control and AS treated groups in male and female F1 rats. In the water-filled T-maze test, pre-test swimming trials in the straight channel revealed that all male and female F1 rats in each group could swim satisfactorily, and no significant changes were observed in the elapsed time to traverse the straight channel. On days 2–4 of the T-maze test, no significant changes were observed in the elapsed time and number of errors in males. In females, the elapsed time and the number of errors on day 2 of the T-maze was significantly lowered at 600 ppm, but there were no significant differences in the elapsed time or number of errors on days 3 and 4 of the T-maze test between control and AS-treated groups (data not shown).

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No compound-related changes in other reproductive/developmental parameters, including developmental neuro-behavioural endpoints.

Dose descriptor:

LOAEL

Generation:

F2

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Body weight was transiently decreased in the 3000 ppm dose group; In F1 and F2 pups, pre-weaning body weight gain was inhibited at 3000 ppm and the liver and spleen weights were decreased at weaning.

Reproductive effects observed:

not specified

Table 1

Reproductive performance of F0 and F1 parental animals

AS (ppm)		0 (control)	120	600	3000
F0 generation
No. of rats (male/female)		24/24	24/24	24/24	24/24
Copulation index (%)^a	Males	91.7	91.7	100	91.7
	Females	95.8	100	100	100
Precoital interval (days)^b		3.2 ± 1.1	3.2 ± 1.8	2.9 ± 1.3	2.8 ± 1.6
Fertility index (%)^c	Males	95.5	90.9	100	95.5
	Females	95.7	91.7	100	95.8
Gestation index (%)^d		100	95.5	95.7	95.7
Gestation length (days)^b		22.4 ± 0.5	22.5 ± 0.6	22.1 ± 0.4	22.3 ± 0.5
Delivery index (%)^{b, e}		94.3 ± 5.6	88.6 ± 21.0	90.7 ± 20.8	92.0 ± 20.5
F1 generation
No. of rats (male/female)		24/24	23/24	24/24	24/24
Copulation index (%)^a	Males	95.8	91.3	95.8	87.5
	Females	100	95.8	100	95.8
Precoital interval (days)^b		3.3 ± 3.2	3.0 ± 2.0	2.7 ± 1.5	2.3 ± 1.1
Fertility index (%)^c	Males	91.3	81.0	91.3	95.2
	Females	91.7	82.6	91.7	91.3
Gestation index (%)^d		100	94.7	100	100
Gestation length (days)^b		22.4 ± 0.5	22.3 ± 0.5	22.2 ± 0.4	22.2 ± 0.4
Delivery index (%)^{b, e}		94.0 ± 9.9	87.5 ± 22.6	91.4 ± 10.7	94.6 ± 6.8

a Copulation index (%) = (no. of animals with successful copulation/no. of animals paired) × 100.

b Values are given as the mean ± S.D.

c Fertility index (%) = (no. of animals that impregnated a female or were pregnant/no. of animals with successful copulation) × 100.

d Gestation index (%) = (no. of females that delivered live pups/no. of pregnant females) × 100.

e Delivery index (%) = (no. of pups delivered/no. of implantations) × 100.

Table 2

Sex ratio, viability and body weight for F1 and F2 pups.

AS (ppm)	0 (control)	120	600	3000
F1 offspring
No. of litters	22	21	22	22
No. of pups delivered^a	13.9 ± 1.7	12.4 ± 4.7	13.1 ± 4.1	13.1 ± 3.4
Sex ratio of pups^b	0.503	0.462	0.513	0.536
Viability index of pups (%)^a
On PND 0^c	100.0 ± 0.0	99.3 ± 2.3	99.7 ± 1.6	99.5 ± 2.4
On PND 4^d	98.7 ± 2.9	95.2 ± 21.8	98.8 ± 2.6	98.0 ± 5.4
On PND 21^e	99.4 ± 2.7	100.0 ± 0.0	100.0 ± 0.0	99.4 ± 2.7
Male pup weight during lactation (g)^a
On PND 0	7.05 ± 0.61	7.25 ± 0.99	6.74 ± 0.69	6.96 ± 0.76
On PND 4	11.04 ± 0.85	11.41 ± 1.99	10.86 ± 1.37	11.00 ± 1.06
On PND 7	18.91 ± 1.29	19.36 ± 2.77	18.59 ± 1.71	18.47 ± 1.35
On PND 14	37.70 ± 2.63	37.97 ± 3.08	37.39 ± 2.59	36.34 ± 2.41
On PND 21	62.48 ± 4.50	62.63 ± 6.14	60.77 ± 4.01	57.34 ± 4.86**
Female pup weight during lactation (g)^a
On PND 0	6.61 ± 0.55	6.89 ± 0.83	6.35 ± 0.57	6.60 ± 0.64
On PND 4	10.46 ± 0.89	11.06 ± 1.71	10.27 ± 1.33	10.43 ± 0.83
On PND 7	18.03 ± 1.27	18.56 ± 2.31	17.69 ± 1.61	17.61 ± 1.21
On PND 14	36.29 ± 2.71	36.94 ± 3.03	35.67 ± 2.60	35.31 ± 2.24
On PND 21	60.17 ± 4.16	60.87 ± 5.68	57.68 ± 4.33	55.60 ± 4.34**
F2 offspring
No. of litters	22	18	22	21
No. of pups delivered^a	13.1 ± 3.6	13.2 ± 3.8	12.6 ± 3.9	14.0 ± 1.9
Sex ratio of pups^b	0.528	0.502	0.536	0.457
Viability index of pups (%)^a
On PND 0^c	99.68 ± 1.51	99.49 ± 2.14	98.42 ± 3.57	98.69 ± 3.60
On PND 4^d	94.72 ± 14.54	98.07 ± 5.45	99.07 ± 3.15	99.01 ± 2.49
On PND 21^e	100.00 ± 0.00	98.61 ± 4.04	100.00 ± 0.00	100.00 ± 0.00
Male pup weight during lactation (g)^a
On PND 0	6.97 ± 0.68	6.92 ± 0.81	6.87 ± 0.74	6.89 ± 0.60
On PND 4	10.73 ± 1.62	10.53 ± 1.27	11.27 ± 1.81	10.52 ± 1.15
On PND 7	17.96 ± 2.05	17.51 ± 2.12	18.83 ± 2.39	17.72 ± 1.60
On PND 14	35.79 ± 3.52	36.18 ± 3.63	37.32 ± 4.15	35.44 ± 2.73
On PND 21	59.61 ± 5.45	59.44 ± 5.67	60.12 ± 7.12	56.36 ± 4.47
Female pup weight during lactation (g)^a
On PND 0	6.66 ± 0.69	6.38 ± 0.78	6.41 ± 0.65	6.50 ± 0.49
On PND 4	10.22 ± 1.63	9.70 ± 1.23	10.36 ± 1.54	9.98 ± 0.91
On PND 7	17.03 ± 1.99	16.36 ± 2.35	17.40 ± 2.18	16.89 ± 1.23
On PND 14	34.82 ± 3.52	34.17 ± 3.58	34.96 ± 4.24	34.01 ± 2.09
On PND 21	57.33 ± 4.90	56.11 ± 5.54	56.41 ± 6.04	54.16 ± 2.82*

a Values are given as the mean ± S.D.

b Sex ratio = total no. of male pups/total no. of pups.

c Viability index on PND 0 (%) = (no. of live pups on PND 0/no. of pups delivered) × 100.

d Viability index on PND 4 (%) = (no. of live pups on PND 4/no. of live pups on PND 0) × 100.

e Viability index on PND 21 (%) = (no. of live pups on PND 21/no. of live pups on PND 4 after cull) × 100.

* Signiﬁcantly different from the control, P < 0.05.

** Signiﬁcantly different from the control, P < 0.01.

Table 3

Absolute and relative organ weight of F1 male and female weanlings

AS (ppm)		0 (control)	120	600	3000
Males
No. of animals		22	20	22	22
Body weight	(g)	90.8 ± 6.9	93.4 ± 10.5	89.7 ± 6.1	79.4 ± 7.5^**
Brain	(g)	1.69 ± 0.06	1.73 ± 0.08	1.72 ± 0.07	1.68 ± 0.05
	(g/100g bw)	1.88 ± 0.13	1.87 ± 0.19	1.92 ± 0.09	2.14 ± 0.17^**
Thymus	(mg)	375 ± 55	384 ± 86	357 ± 58	305 ± 51^**
	(mg/100 g bw)	414 ± 56	409 ± 64	398 ± 59	383 ± 36
Liver	(g)	4.33 ± 0.43	4.40 ± 0.60	4.22 ± 0.45	3.49 ± 0.53^**
	(g/100 g bw)	4.77 ± 0.30	4.71 ± 0.33	4.70 ± 0.27	4.37 ± 0.30^**
Kidney^a	(g)	1.06 ± 0.09	1.09 ± 0.14	1.03 ± 0.11	0.95 ± 0.13^**
	(g/100 g bw)	1.17 ± 0.06	1.16 ± 0.07	1.15 ± 0.08	1.20 ± 0.07
Spleen	(mg)	394 ± 49	410 ± 68	388 ± 74	301 ± 43**
	(mg/100 g bw)	436 ± 63	437 ± 40	432 ± 73	379 ± 37**
Testisa	(mg)	596 ± 65	583 ± 67	569 ± 65	539 ± 51*
	(mg/100 g bw)	657 ± 64	626 ± 49	635 ± 64	682 ± 58
Epididymis^a	(mg)	81.8 ± 8.6	76.8 ± 10.9	76.5 ± 8.4	72.0 ± 9.9^**
	(mg/100 g bw)	90.4 ± 10.3	82.0 ± 6.1	85.4 ± 8.4	91.5 ± 14.6
Females
No. of animals		22	20	22	21
Body weight	(g)	84.3 ± 6.3	85.9 ± 9.2	80.5 ± 7.0	75.8 ± 6.4^**
Brain	(g)	1.64 ± 0.06	1.66 ± 0.06	1.63 ± 0.05	1.63 ± 0.07
	(g/100 g bw)	1.96 ± 0.12	1.95 ± 0.18	2.04 ± 0.17	2.16 ± 0.14^**
Thymus	(mg)	383 ± 66	373 ± 74	345 ± 46	313 ± 33^**
	(mg/100 g bw)	453 ± 63	433 ± 64	429 ± 57	415 ± 41
Liver	(g)	3.83 ± 0.47	3.92 ± 0.48	3.61 ± 0.35	3.24 ± 0.34^**
	(g/100 g bw)	4.53 ± 0.30	4.57 ± 0.31	4.48 ± 0.30	4.27 ± 0.25^*
Kidneya	(g)	0.99 ± 0.11	0.99 ± 0.09	0.93 ± 0.10	0.93 ± 0.10
	(g/100 g bw)	1.17 ± 0.08	1.15 ± 0.07	1.15 ± 0.09	1.23 ± 0.09
Spleen	(mg)	337 ± 62	356 ± 55	341 ± 64	292 ± 43^*
	(mg/100 g bw)	400 ± 67	415 ± 44	422 ± 53	386 ± 47
Ovarya	(mg)	25.3 ± 4.8	25.3 ± 3.8	22.5 ± 4.6	24.7 ± 3.2
	(mg/100 g bw)	30.1 ± 5.1	29.7 ± 5.0	27.9 ± 5.0	32.5 ± 4.2
Uterus	(mg)	70.6 ± 16.6	74.2 ± 32.0	59.2 ± 11.9^*	55.4 ± 13.4^**
	(mg/100 g bw)	83.8 ± 19.2	85.5 ± 32.4	73.3 ± 11.9	73.3 ± 18.0

Values are given as the mean ± S.D.

a Values represent the total weights of the organs on both sides.

* Signiﬁcantly different from the control, P < 0.05.

** Signiﬁcantly different from the control, P < 0.01.

Table 4

Absolute and relative organ weight of F2 male and female weanlings

AS (ppm)		0 (control)	120	600	3000
Males
No. of animals		21	18	22	21
Body weight	(g)	87.7 ± 5.8	89.0 ± 8.7	87.0 ± 9.6	79.2 ± 6.8^**
Brain	(g)	1.66 ± 0.05	1.69 ± 0.06	1.70 ± 0.06	1.67 ± 0.06
	(g/100 g bw)	1.90 ± 0.13	1.91 ± 0.17	1.97 ± 0.16	2.13 ± 0.17^**
Thymus	(mg)	382 ± 50	348 ± 49	357 ± 66	305 ± 36^**
	(mg/100 g bw)	439 ± 70	392 ± 52^*	411 ± 57	386 ± 40^**
Liver	(g)	3.93 ± 0.37	4.04 ± 0.64	3.91 ± 0.39	3.45 ± 0.41^**
	(g/100 g bw)	4.49 ± 0.34	4.52 ± 0.44	4.50 ± 0.24	4.36 ± 0.23
Kidney^a	(g)	1.02 ± 0.09	1.01 ± 0.13	0.99 ± 0.13	0.94 ± 0.10
	(g/100 g bw)	1.16 ± 0.08	1.14 ± 0.06	1.14 ± 0.07	1.19 ± 0.06
Spleen	(mg)	368 ± 54	381 ± 62	361 ± 49	296 ± 48^**
	(mg/100 g bw)	421 ± 64	427 ± 50	416 ± 48	372 ± 42^**
Testis^a	(mg)	559 ± 67	549 ± 98	543 ± 77	534 ± 54
	(mg/100 g bw)	637 ± 60	615 ± 81	624 ± 47	680 ± 92
Epididymis^a	(mg)	75.3 ± 6.9	78.3 ± 8.8	75.1 ± 10.7	70.5 ± 5.7^*
	(mg/100 g bw)	86.1 ± 8.3	88.4 ± 9.0	86.5 ± 9.0	89.4 ± 8.2
Females
No. of animals		22	18	21	21
Body weight	(g)	80.8 ± 6.0	80.0 ± 7.2	80.8 ± 9.1	73.8 ± 4.4^**
Brain	(g)	1.60 ± 0.06	1.61 ± 0.05	1.64 ± 0.05*	1.61 ± 0.04
	(g/100 g bw)	1.99 ± 0.14	2.03 ± 0.16	2.05 ± 0.20	2.19 ± 0.15^**
Thymus	(mg)	337 ± 45	364 ± 36	347 ± 49	312 ± 37
	(mg/100 g bw)	419 ± 61	457 ± 50	431 ± 47	424 ± 54
Liver	(g)	3.56 ± 0.35	3.61 ± 0.39	3.61 ± 0.48	3.07 ± 0.26^**
	(g/100 g bw)	4.41 ± 0.21	4.51 ± 0.26	4.47 ± 0.26	4.17 ± 0.29^**
Kidney^a	(g)	0.95 ± 0.07	0.93 ± 0.10	0.92 ± 0.10	0.88 ± 0.08
	(g/100 g bw)	1.18 ± 0.08	1.16 ± 0.09	1.14 ± 0.06	1.20 ± 0.07
Spleen	(mg)	320.9 ± 46.7	331.8 ± 59.3	331.3 ± 57.1	269.9 ± 55.2^**
	(mg/100 g bw)	398.4 ± 59.0	414.8 ± 64.3	409.0 ± 42.2	365.0 ± 67.4
Ovary^a	(mg)	23.9 ± 3.7	22.8 ± 3.6	23.2 ± 3.5	20.2 ± 2.3^**
	(mg/100 g bw)	29.7 ± 4.9	28.8 ± 5.6	29.0 ± 4.7	27.5 ± 3.5
Uterus	(mg)	60.5 ± 17.0	63.8 ± 18.4	65.0 ± 41.7	49.3 ± 11.6^*
	(mg/100 g bw)	74.6 ± 19.2	79.3 ± 19.3	78.7 ± 40.4	67.0 ± 16.2

Values are given as the mean ± S.D.

a Values represent the total weights of the organs on both sides.

* Signiﬁcantly different from the control, P < 0.05.

** Signiﬁcantly different from the control, P < 0.01.

Conclusions:

In a two-generation study rats were continuously exposed to Aluminum sulfate via drinking water. The resulting NOAEL was 600 ppm (41.0 mg/kg bw/day) and the LOAEL was 3000 ppm (188.0 mg/kg bw/day).

Executive summary:

In a 2-generation reproduction study according to OECD 416, Aluminium sulfate (98.5 % purity) was administered to Crl:CD(SD) rats in water at dose levels of 0, 120, 600 and 3000 ppm (0, 8.6, 41.0 and 188 mg/kg bw/day).

No compound related effects were noted in the main categories of systemic or reproductive toxicity evaluated, except a transiently decreased body weight in the 3000 ppm dose group. Moreover, in F1 and F2 pups, the preweaning body weight gain was inhibited at 3000 ppm and the liver and spleen weights were decreased at weaning. At this dose, vaginal opening was slightly delayed. There were no compound-related changes in other reproductive/developmental parameters, including developmental neurobehavioral endpoints.

Based on these findings, the LOAEL is 3000 ppm (188 mg/kg bw/day) and the NOAEL for parental systemic toxicity and reproductive/developmental toxicity is 600 ppm (41 mg/kg bw/day), which equals a daily aluminium intake of 8.06 mg Al/kg bw/day.

This study is acceptable and satisfies the guideline requirement for a 2-generation reproductive study according to OECD 416 in rat.

This information is used in a read-across approach in the assessment of the target substance. For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

41 mg/kg bw/day

Study duration:

chronic

Species:

rat

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Since no data of aluminium nitride (target substance) are available, the potential of aluminium nitride is assessed by its dissolution products aluminium and ammonium ions. Ammonium is an endogenous compound, formed at concentrations of 79 mmol/kg bw/day. Therefore, the nitride component of aluminium nitride is considered to be of minor impact and negligible for the assessment of the toxicity to reproduction. The potential of aluminium nitride is thus based on the aluminium component and assessed by read across with soluble aluminium salts, mainly aluminium ammonium sulfate and aluminium sulfate. Due to a lower water solubility of aluminium nitride compared to the source substances the resulting bioavailability and subsequent toxicity potential would also be expected to be lower. Therefore, the read-across to the source substances aluminium ammonium sulfate and aluminium sulfate is adequately protective. Details on the read-across rational are provided in section 13.

In a two-generation reproduction study aluminium sulfate (98.5%) was continuously given orally via drinking water to groups of Crl:CD(SD) rats according to OECD guideline 416 at concentrations of 120, 600 and 3000 ppm (nominal in water). In the 3000 pm dose group the F1 and F2 animals showed a statistically significant decrease in body weight and in certain organs in comparison to the untreated control. Gross pathology of delivered pups (F1/F2) revealed no significant difference in the incidence between control and treatment groups. Moreover, no dose related histopathological changes in the male and female F1 and F2 weanlings were observed.

Based on the results, a NOAEL of 600 ppm was determined. Based on water consumption and body weight the mean daily test item intake of the 600 ppm dose group was calculated as 41 mg/kg bw/day (which equals an ingested aluminium dose of 8.06 mg/kg bw/day).

In a second two-generation reproduction study (OECD 416) aluminium ammonium sulfate (99.5 % purity ) was continuously given to groups of Crl:CD(SD) rats via drinking water at levels of 0, 50, 500 and 5000 ppm. In conclusion, the NOAEL of aluminium ammonium sulfate for two-generation reproductive/developmental toxicity was considered to be 500 ppm in rats, primarily based on the depression of preweaning body weight gain. Considering the aluminium content in the basal diet, the total ingested dose of aluminium from drinking water and food in this 500 ppm group was calculated to be 5.35 mg Al/kg bw/day.

In a developmental toxicity study Al(OH)3 was administered to pregnant Wistar rats in water, split into 2 gavages per day at dose levels of 0, of 192, 378 and 768 mg/kg/ from days 6 through 15 of gestation. No treatment related maternal and/or developmental effects were noted. The maternal/developmental NOAEL was determined with 768 mg/kg/day.

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Details on maternal toxic effects:

Maternal toxic effects: no effects

Details on maternal toxic effects:
No maternal deaths or signs of toxicity were observed during the study.

Dose descriptor:

NOAEL

Effect level:

768 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

No embryotoxic effects were observed during the study.

Dose descriptor:

NOAEL

Effect level:

768 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Statistically significant decreases in maternal food consumption were found during the treatment period and during the total study period and were not considered treatment-related toxic effects since those differences were sporadic and not dose related. Maternal stress during the gavage administration (twice a day) may have been responsible for the decreases in food consumption. The concentrations of Al in maternal liver, maternal brain, maternal bone and placenta as well as in whole-body fetuses did not differ significantly between the control and Al(OH)3-treated groups.

Conclusions:

Administration of Aluminum hydroxide (twice a day by gavage) from day 6 to 15 of gestation resulted in a NOAEL of 768 mg/kg/day for Al(OH)3, which corresponds to a Aluminum dose of 207 mg/kg/day.

Executive summary:

In a developmental toxicity study (similar to OECD 414), Aluminum hydroxide was administered to 20 pregnant female Wistar rats per dose group. The dosages were split into 2 gavages per day resulting in dose levels of 0,192, 378 and 768 mg/kg body weight from days 6 to 15 of gestation.

No treatment-related maternal and developmental signs of toxicity were observed at any dose level. Thus, the maternal and the developmental NOAEL is 768 mg/kg/day.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rat, with deviation in the exposure period.

This information is used in a read-across approach in the assessment of the target substance. For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

768 mg/kg bw/day

Study duration:

subacute

Species:

rat

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

In a developmental toxicity study Al(OH)3 was administered to pregnant Wistar rats in water, split into 2 gavages per day at dose levels of 0, of 192, 378 and 768 mg/kg/ from days 6 through 15 of gestation. No treatment related maternal and/or developmental effects were noted. The maternal/developmental NOAEL was determined with 768 mg/kg/day, which corresponds to an aluminium dose of 207 mg/kg/day.

Aluminium hydroxide is an acceptable read-across partner as aluminium is considered to be the toxicological relevant part. Details on the read-across rational are provided in section 13.

No adverse effects on reproduction and pre-natal development were reported in the selected studies. Based on the available data aluminium nitride does not warrant classification for reproductive toxicity.