ALFERROCK

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No reproductive toxicity data is available for the target substance.

In the absence of substance specific data for Alferrock, a conservative approach to hazard evaluation is to assume that aluminium released from Alferrock shows the same systemic hazards as other aluminium compounds with similar or higher bioavailability. Read-across to aluminium compounds with a high release of aluminium ions (soluble Al salts) is considered to represent a worst-case approach that overestimates toxicological properties of the target compound. For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

 

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.

Link to relevant study records

Reference

Reference 1

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 report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

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

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

see below

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

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:

no effects observed

Description (incidence and severity):

see below

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption was significantly lower during week 1 of dosing in F0 males of the 5000 ppm group and in F0 females of the 500 and 5000 ppm groups. In 5000 ppm-treated F0
and F1 females, there were also significant decreases in food consumption in the 2nd and 3rd weeks of lactation.
Body weight was significantly lower in the 2nd week of dosing in both sexes of F0 rats and on day 21 of lactation in F0 females at 5000 ppm. In the 5000 ppm group, the body weight of F1 males and females was significantly lower in the first 2 and 3 weeks of dosing, respectively.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Significant changes were observed throughout or almost throughout the dosing period in F0 males of all AAS-treated groups, and in F0 females and F1 males and females in the 500 and 5000 ppm groups. Significant decreases in water consumption were also found during weeks 1, 9 and 10 of dosing, week 1 of gestation and week 1 of lactation in 50 ppm-treated F0 females and during weeks 4 and 8–10 of dosing in 50 ppm-treated F1 females.
Based on water consumption and body weight, daily AAS 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 AAS-treated groups.

Calculated mean AAS intake during the whole period was:
3.78, 33.5 and 305 mg/kg bw/day in F0 males,
6.52, 58.6 and 500 mg/kg bw/day in F0 females,
4.59, 41.8 and 372 mg/kg bw/day in F1 males, and
6.65, 61.9 and 517 mg/kg bw/day in F1 females,
for the 50, 500 and 5000 ppm groups, respectively.

Considering aluminium content in the basal diet, dietary aluminium exposure of F0 and F1 animals was estimated from the food consumption and body weight in the control and AAS-treated groups. Average aluminium intake from drinking water and food combined was calculated to be:
1.56, 1.98, 5.35 and 36.3 mg Al/kg bw/day in F0 males,
2.20, 2.89, 8.81 and 59.0 mg Al/kg bw/day in F0 females,
1.83, 2.35, 6.57 and 44.2 mg Al/kg bw/day in F1 males, and
2.39, 3.10, 9.36 and 61.1 mg Al/kg bw/day in F1 females
for control through high-dose groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
During the premating period, a few AAS-treated F0 and F1 female rats had persistent diestrus; however, the incidence of females with normal estrous cycles (4–5 days) was not changed significantly compared with the control. There were no significant differences in the estrous cycle between control and AAS-treated groups.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Sperm analysis of schedule-sacrificed F0 and F1 adults revealed no significant differences in the number of testis sperm and cauda epididymal 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 AAS-treated groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Although some animals failed to copulate, impregnate or deliver live pups, no significant changes were found in the copulation, fertility or gestation index between the control and AAS-treated groups in F0 and F1 generations. There were also no significant differences in the precoital interval and gestation length in either generation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In F0 females in the 500 and 5000 ppm groups and in F1 males and females in the 5000 ppm group, relative kidney weight was increased significantly. A significant decrease in the absolute weight of the pituitary gland was found in F0 females and in F1 males and females at 5000 ppm. In F1 females, there was also a significant decrease in the absolute thymus weight at 5000 ppm. Further, significant decreases were found in the relative weight of the seminal vesicle in 50 ppm-treated F1 males and in the absolute brain weight in 500 ppm-treated F1 females, but no dose-dependency was found in these changes.

Dose descriptor:

NOAEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: 500 ppm AAS equals 5.35 mg Al/kg bw/d

Dose descriptor:

LOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: decreased body weight

Clinical signs:

no effects observed

Mortality:

no mortality observed

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:

no effects observed

Description (incidence and severity):

see below

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption was significantly lower during week 1 of dosing in F0 males of the 5000 ppm group and in F0 females of the 500 and 5000 ppm groups. In 5000 ppm-treated F0
and F1 females, there were also significant decreases in food consumption in the 2nd and 3rd weeks of lactation.
Body weight was significantly lower in the 2nd week of dosing in both sexes of F0 rats and on day 21 of lactation in F0 females at 5000 ppm. In the 5000 ppm group, the body weight of F1 males and females was significantly lower in the first 2 and 3 weeks of dosing, respectively.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Significant changes were observed throughout or almost throughout the dosing period in F0 males of all AAS-treated groups, and in F0 females and F1 males and females in the 500 and 5000 ppm groups. Significant decreases in water consumption were also found during weeks 1, 9 and 10 of dosing, week 1 of gestation and week 1 of lactation in 50 ppm-treated F0 females and during weeks 4 and 8–10 of dosing in 50 ppm-treated F1 females.
Based on water consumption and body weight, daily AAS 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 AAS-treated groups.

Calculated mean AAS intake during the whole period was:
3.78, 33.5 and 305 mg/kg bw/day in F0 males,
6.52, 58.6 and 500 mg/kg bw/day in F0 females,
4.59, 41.8 and 372 mg/kg bw/day in F1 males, and
6.65, 61.9 and 517 mg/kg bw/day in F1 females,
for the 50, 500 and 5000 ppm groups, respectively.

Considering aluminium content in the basal diet, dietary aluminium exposure of F0 and F1 animals was estimated from the food consumption and body weight in the control and AAS-treated groups. Average aluminium intake from drinking water and food combined was calculated to be:
1.56, 1.98, 5.35 and 36.3 mg Al/kg bw/day in F0 males,
2.20, 2.89, 8.81 and 59.0 mg Al/kg bw/day in F0 females,
1.83, 2.35, 6.57 and 44.2 mg Al/kg bw/day in F1 males, and
2.39, 3.10, 9.36 and 61.1 mg Al/kg bw/day in F1 females
for control through high-dose groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
During the premating period, a few AAS-treated F0 and F1 female rats had persistent diestrus; however, the incidence of females with normal estrous cycles (4–5 days) was not changed significantly compared with the control. There were no significant differences in the estrous cycle between control and AAS-treated groups.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Sperm analysis of schedule-sacrificed F0 and F1 adults revealed no significant differences in the number of testis sperm and cauda epididymal 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 AAS-treated groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Although some animals failed to copulate, impregnate or deliver live pups, no significant changes were found in the copulation, fertility or gestation index between the control and AAS-treated groups in F0 and F1 generations. There were also no significant differences in the precoital interval and gestation length in either generation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In F0 females in the 500 and 5000 ppm groups and in F1 males and females in the 5000 ppm group, relative kidney weight was increased significantly. A significant decrease in the absolute weight of the pituitary gland was found in F0 females and in F1 males and females at 5000 ppm. In F1 females, there was also a significant decrease in the absolute thymus weight at 5000 ppm. Further, significant decreases were found in the relative weight of the seminal vesicle in 50 ppm-treated F1 males and in the absolute brain weight in 500 ppm-treated F1 females, but no dose-dependency was found in these changes.

Dose descriptor:

NOAEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: 500 ppm AAS equals 5.35 mg Al/kg bw/d

Dose descriptor:

LOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

effects observed, treatment-related

Anogenital distance (AGD):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
No significant changes were found in the number of implantations or pups delivered, delivery index, sex ratio of pups and the viability index during the preweaning period in either generation.

BODY WEIGHT (OFFSPRING)
While there were no significant differences in birth weight between the control and AAS-treated groups, the body weight of F1 males on PND 21 and ofF1 females on PNDs 14 and 21 was significantly lower in the 5000 ppm group than in the control. A similar decreasing trend was found in the body weight of male and female F2 pups around the time of weaning in the highest dose group, although no statistical significance was
found.

SEXUAL MATURATION (OFFSPRING)
In F1 female animals, vaginal opening was significantly delayed at 5000 ppm (32.3 ± 1.8 days of age, compared with 30.2 ± 2.1 days of age in controls, P 6 0.01). Body weight at the time of attainment was not significantly, but was slightly heavier in this 5000 ppm group (122.0 ± 15.7 g, compared with 115.8 ± 12.6 g in control). There were no significant differences in age at preputial separation or body weight at the time of completion in F1 males between control and AAS-treated groups.

ORGAN WEIGHTS (OFFSPRING)
In F1 and F2 weanlings, 5000 ppm-treated males and females had significantly lower body weights, and the absolute and relative weights of the spleen in both sexes and of the thymus in males were significantly decreased in this 5000 ppm group. A decrease in the absolute thymus weight was also observed in F1 females given 500 and 5000 ppm and in F2 females given 5000 ppm, but there were no significant changes in relative weight in F1 or F2 females. The absolute liver weight was significantly decreased in F1 and F2 males and females, accompanied with a decrease in the relative weight in F1 males and F2 females in the 5000 ppm group. The relative weights of the brain and kidney were increased significantly in F1 and F2 males and females given 5000 ppm. Further, a significant decrease in the absolute weight of the kidney, adrenal, testis, epididymis, ovary and uterus was found at 500 and/or 5000 ppm.

GROSS PATHOLOGY (OFFSPRING)
During the preweaning period, external gross examination revealed microphthalmia, a rudimentary tail, trauma and scab on right hindlimb and crushing of incisor/malocclusion in a few F1 pups in control and AAS-treated groups; however, there were no significant differences in incidence between the control and AAS treated groups.
No gross abnormalities were found in any F2 pups.

HISTOPATHOLOGY (OFFSPRING)
No dose-related changes were found in the histopathology of the liver and spleen in both
sexes and of the thymus in males in either generation.

OTHER FINDINGS (OFFSPRING)
In F1 and F2 pups, there were no significant differences in the completion rate of pinna unfolding, the age at completion of incisor eruption and eye opening, and AGD and AGD per cube root of the body weight ratio between the control and AAS-treated groups. All male and female F1 and F2 pups in all groups achieved the surface righting reflex on PND 5, negative geotaxis reflex on PND 8 and mid-air righting reflex on PND 18, and no significant changes were found in the response time of surface righting
and negative geotaxis reflex.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: 500 ppm AAS equals 5.35 mg Al/kg bw/d

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Anogenital distance (AGD):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
No significant changes were found in the number of implantations or pups delivered, delivery index, sex ratio of pups and the viability index during the preweaning period in either generation.

BODY WEIGHT (OFFSPRING)
While there were no significant differences in birth weight between the control and AAS-treated groups, the body weight of F1 males on PND 21 and ofF1 females on PNDs 14 and 21 was significantly lower in the 5000 ppm group than in the control. A similar decreasing trend was found in the body weight of male and female F2 pups around the time of weaning in the highest dose group, although no statistical significance was
found.

SEXUAL MATURATION (OFFSPRING)
In F1 female animals, vaginal opening was significantly delayed at 5000 ppm (32.3 ± 1.8 days of age, compared with 30.2 ± 2.1 days of age in controls, P 6 0.01). Body weight at the time of attainment was not significantly, but was slightly heavier in this 5000 ppm group (122.0 ± 15.7 g, compared with 115.8 ± 12.6 g in control). There were no significant differences in age at preputial separation or body weight at the time of completion in F1 males between control and AAS-treated groups.

ORGAN WEIGHTS (OFFSPRING)
In F1 and F2 weanlings, 5000 ppm-treated males and females had significantly lower body weights, and the absolute and relative weights of the spleen in both sexes and of the thymus in males were significantly decreased in this 5000 ppm group. A decrease in the absolute thymus weight was also observed in F1 females given 500 and 5000 ppm and in F2 females given 5000 ppm, but there were no significant changes in relative weight in F1 or F2 females. The absolute liver weight was significantly decreased in F1 and F2 males and females, accompanied with a decrease in the relative weight in F1 males and F2 females in the 5000 ppm group. The relative weights of the brain and kidney were increased significantly in F1 and F2 males and females given 5000 ppm. Further, a significant decrease in the absolute weight of the kidney, adrenal, testis, epididymis, ovary and uterus was found at 500 and/or 5000 ppm.

GROSS PATHOLOGY (OFFSPRING)
During the preweaning period, external gross examination revealed microphthalmia, a rudimentary tail, trauma and scab on right hindlimb and crushing of incisor/malocclusion in a few F1 pups in control and AAS-treated groups; however, there were no significant differences in incidence between the control and AAS treated groups.
No gross abnormalities were found in any F2 pups.

HISTOPATHOLOGY (OFFSPRING)
No dose-related changes were found in the histopathology of the liver and spleen in both
sexes and of the thymus in males in either generation.

OTHER FINDINGS (OFFSPRING)
In F1 and F2 pups, there were no significant differences in the completion rate of pinna unfolding, the age at completion of incisor eruption and eye opening, and AGD and AGD per cube root of the body weight ratio between the control and AAS-treated groups. All male and female F1 and F2 pups in all groups achieved the surface righting reflex on PND 5, negative geotaxis reflex on PND 8 and mid-air righting reflex on PND 18, and no significant changes were found in the response time of surface righting
and negative geotaxis reflex.

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

500 ppm

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: 500 ppm AAS equals 5.35 mg Al/kg b.w.

Dose descriptor:

LOAEL

Generation:

F2

Effect level:

5 000 ppm

Sex:

male/female

Basis for effect level:

body weight and weight gain

Reproductive effects observed:

not specified

Table 1         Reproductive performance of F0 and F1 parental animals.
AAS (ppm)		0 (control)	50	500	5000
F0 generation
No. of rats (male/female)		24/24	24/24	24/24	24/24
Copulation index (%)^a	males	100	95.8	91.7	100
	females	100	100	100	100
Precoital inverval (days)^b		2.2±1.0	2.5±1.6	2.3±1.2	2.8±1.6
Fertility index (%)^c	males	100	91.3	100	100
	females	100	87.5	100	100
Gestation index (%)^d		100	100	100	100
Gestation length (days)^b		22.3±0.5	22.4±0.6	22.3±0.5	22.4±0.5
F1 generation
No. of rats (male/female)		24/24	24/24	23/24	24/24
Copulation index (%)^a	males	91.7	91.7	91.3	95.8
	females	100	95.8	100	100
Precoital inverval (days)^b		2.7±1.8	3.0±2.1	3.3±2.4	3.1±1.3
Fertility index (%)^c	males	90.9	77.3	95.2	100
	females	91.7	78.3	95.8	95.8
Gestation index (%)^d		100	100	95.7	100
Gestation length (days)^b		22.3±0.5	22.3±0.5	22.2±0.4	22.2±0.4
^aCopulation index (%) = (No. of animals with successful copulation/No. of animals paired) x 100.
^bValues are given as the mean ± S.D.
^cFertility index (%) = (No. of animals that impregnated a female or were pregnant/No. of animals with successful copulation) x 100.
^dGestation index (%) = (No. of females that delivered live pups/No. of pregnant females) x 100.

Table 2
Developmental findings for F1 and F2 offsprings.
AAS (ppm)	0 (control)	50	500	5000
F0 parents/F1 offspring
No. of F0 pregnant females	24	21	24	24
No. of implantations^a	14.7 ± 3.1	14.3 ± 2.1	15.0 ± 3.3	15.1 ± 1.5
No. of litters	24	21	24	24
No. of pups delivered^a	13.6 ± 3.1	13.5 ± 2.5	13.8 ± 3.1	14.4 ± 1.6
Delivery index (%)^a,b	92.4 ± 8.0	94.2 ± 10.3	92.3 ± 7.8	95.4 ± 5.4
Sex ratio of pups^c	0.509	0.493	0.476	0.487
Viability index of pups (%)^a
On PND 0^d	99.5 ± 2.7	99.0 ± 2.4	99.5 ± 1.7	99.2 ± 2.3
On PND 4^e	98.3 ± 5.0	98.0 ± 5.4	95.6 ± 20.4	99.2 ± 2.3
On PND 21^f	100.0 ± 0.0	100.0 ± 0.0	100.0 ± 0.0	100.0 ± 0.0
Male pup weight during lactation (g)^a
On PND 0	6.93 ± 0.66	6.96 ± 0.68	6.91 ± 0.48	6.90 ± 0.69
On PND 4	11.13 ± 1.88	10.84 ± 1.47	10.72 ± 0.94	10.68 ± 1.33
On PND 7	19.14 ± 2.30	18.86 ± 2.30	18.71 ± 1.51	18.49 ± 1.70
On PND 14	38.45 ± 3.57	38.32 ± 3.96	37.88 ± 2.31	36.51 ± 2.20
On PND 21	63.83 ± 5.93	62.59 ± 7.09	61.71 ± 4.94	58.67 ± 3.91**
Female pup weight during lactation (g)^a
On PND 0	6.66 ± 0.82	6.57 ± 0.61	6.58 ± 0.57	6.43 ± 0.63
On PND 4	10.70 ± 2.02	10.34 ± 1.25	10.22 ± 1.13	10.13 ± 1.28
On PND 7	18.40 ± 2.49	17.96 ± 2.02	17.97 ± 1.74	17.38 ± 1.79
On PND 14	37.23 ± 3.65	36.97 ± 3.30	36.59 ± 2.74	35.07 ± 2.35*
On PND 21	61.65 ± 6.05	60.03 ± 5.55	59.34 ± 5.22	56.13 ± 4.07**
F1 parents/F2 offspring
No. of F1 parent females	22	18	23	23
No. of implantations^a	15.0 ± 1.6	14.7 ± 1.7	14.7 ± 3.5	14.1 ± 2.2
No. of litters	22	18	22	23
No. of pups delivered^a	13.9 ± 1.8	13.7 ± 2.4	14.0 ± 3.8	13.5 ± 2.1
Delivery index (%)^a,b	92.7 ± 9.4	93.0 ± 11.2	90.9 ± 20.4	95.6 ± 5.7
Sex ratio of pups^c	0.435	0.500	0.492	0.506
Viability index of pups (%)^a
On PND 0^d	98.3 ± 4.5	97.6 ± 4.2	98.9 ± 3.3	99.7 ± 1.5
On PND 4^e	97.9 ± 6.0	99.4 ± 2.4	99.4 ± 1.9	99.0 ± 2.9
On PND 21^f	99.4 ± 2.7	100.0 ± 0.0	100.0 ± 0.0	100.0 ± 0.0
Male pup weight during lactation (g)^a
On PND 0	6.97 ± 0.62	7.03 ± 0.65	6.89 ± 0.53	6.97 ± 0.75
On PND 4	10.64 ± 1.62	11.31 ± 1.22	10.95 ± 1.32	11.16 ± 1.87
On PND 7	17.97 ± 2.18	19.19 ± 1.73	18.82 ± 1.90	18.42 ± 2.39
On PND 14	36.89 ± 3.26	38.99 ± 3.14	38.28 ± 3.26	36.40 ± 3.67
On PND 21	61.07 ± 6.06	64.40 ± 5.58	63.20 ± 5.51	58.65 ± 5.90
Female pup weight during lactation (g)^a
On PND 0	6.46 ± 0.47	6.68 ± 0.67	6.54 ± 0.50	6.51 ± 0.63
On PND 4	9.91 ± 1.26	10.62 ± 1.18	10.30 ± 1.20	10.59 ± 1.72
On PND 7	17.15 ± 2.06	18.28 ± 1.77	17.73 ± 1.68	17.58 ± 2.34
On PND 14	35.58 ± 3.00	37.47 ± 2.74	36.65 ± 2.69	35.20 ± 3.44
On PND 21	58.47 ± 5.33	61.83 ± 4.40	60.05 ± 3.82	56.72 ± 5.39

* Significantly different from the control, P < 0.05.

** Significantly different from the control, P < 0.01

a Values are given as the mean ± S.D.

b Delivery index (%) = (No. of pups delivered/No. of implantations) x 100.

c Sex ratio = total No. of male pups/total No. of pups.

d Viability index on PND 0 (%) = (No. of live pups on PND 0/No. of pups delivered) x 100.

e Viability index on PND 4 (%) = (No. of live pups on PND 4/No. of live pups on PND 0) x 100.

f Viability index on PND 21 (%) = (No. of live pups on PND 21/No. of live pups on PND 4 after cull) x 100.

Table 3
Absolute and relative organ weight of F1 male and female weanlings.
AAS (ppm)		0 (control)	50	500	5000
Males
No. of animals		24	20	23	24
Body weight	(g)	94.1 ± 9.1	90.8 ± 10.7	91.3 ± 9.8	80.9 ± 7.5**
Brain	(g)	1.72 ± 0.08	1.71 ± 0.07	1.70 ± 0.06	1.68 ± 0.07
	(g/100 g b.w.)	1.84 ± 0.16	1.90 ± 0.17	1.88 ± 0.16	2.09 ± 0.15**
Thymus	(mg)	392 ± 67	373 ± 72	360 ± 57	301 ± 48**
	(mg/100 g b.w.)	417 ± 61	411 ± 55	396 ± 61	372 ± 52*
Liver	(g)	4.32 ± 0.54	4.15 ± 0.55	4.12 ± 0.53	3.52 ± 0.43**
	(g/100 g b.w.)	4.58 ± 0.29	4.57 ± 0.17	4.51 ± 0.27	4.34 ± 0.25**
Kidney^a	(g)	1.08 ± 0.13	1.04 ± 0.14	1.05 ± 0.10	0.98 ± 0.10*
	(g/100 g b.w.)	1.15 ± 0.10	1.15 ± 0.08	1.15 ± 0.06	1.21 ± 0.08*
Spleen	(mg)	421 ± 75	399 ± 66	403 ± 91	292 ± 49**
	(mg/100 g b.w.)	447 ± 64	441 ± 60	439 ± 78	361 ± 43**
Adrenal^a	(mg)	26.4 ± 3.4	24.5 ± 2.7	25.5 ± 3.2	24.0 ± 3.4*
	(mg/100 g b.w.)	28.2 ± 3.6	27.2 ± 2.9	28.0 ± 3.0	29.8 ± 3.5
Testis^a	(mg)	591 ± 69	571 ± 74	573 ± 72	532 ± 78*
	(mg/100 g b.w.)	628 ± 38	630 ± 41	628 ± 49	656 ± 61
Epididymis^a	(mg)	80.7 ± 9.3	76.2 ± 10.7	78.9 ± 10.0	67.8 ± 9.9**
	(mg/100 g b.w.)	86.0 ± 8.1	84.3 ± 10.4	86.6 ± 8.3	84.2 ± 11.6
Females
No. of animals		24	21	23	24
Body weight	(g)	87.0 ± 7.2	85.5 ± 7.6	83.3 ± 7.1	76.2 ± 7.0**
Brain	(g)	1.68 ± 0.12	1.64 ± 0.06	1.65 ± 0.06	1.62 ± 0.06
	(g/100 g b.w.)	1.93 ± 0.16	1.93 ± 0.16	1.99 ± 0.14	2.14 ± 0.16**
Thymus	(mg)	382 ± 58	365 ± 48	342 ± 51*	316 ± 41**
	(mg/100 g b.w.)	437 ± 46	429 ± 56	411 ± 54	416 ± 54
Liver	(g)	3.79 ± 0.38	3.80 ± 0.39	3.73 ± 0.42	3.28 ± 0.43**
	(g/100 g b.w.)	4.36 ± 0.38	4.45 ± 0.28	4.48 ± 0.31	4.30 ± 0.28
Kidney^a	(g)	0.98 ± 0.10	0.97 ± 0.11	0.96 ± 0.09	0.93 ± 0.08
	(g/100 g b.w.)	1.13 ± 0.08	1.14 ± 0.06	1.15 ± 0.05	1.22 ± 0.07**
Spleen	(mg)	362 ± 63	351 ± 44	356 ± 59	272 ± 47**
	(mg/100 g b.w.)	416 ± 72	412 ± 49	428 ± 63	356 ± 46**
Adrenal^a	(mg)	25.5 ± 3.9	23.6 ± 3.0	22.7 ± 3.0**	22.3 ± 2.6**
	(mg/100 g b.w.)	29.4 ± 4.0	27.8 ± 3.8	27.3 ± 3.5	29.4 ± 3.1
Ovary^a	(mg)	24.6 ± 4.5	24.6 ± 4.4	23.8 ± 2.9	22.0 ± 4.0
	(mg/100 g b.w.)	28.2 ± 4.6	29.0 ± 4.6	28.9 ± 4.6	29.2 ± 6.4
Uterus	(mg)	67.3 ± 15.3	66.4 ± 21.4	64.6 ± 15.9	50.4 ± 10.9**
	(mg/100 g b.w.)	77.3 ± 16.6	77.1 ± 20.5	77.2 ± 15.8	66.2 ± 12.8

Values are given as the mean ± S.D.

* Significantly different from the control, P < 0.05.

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

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

Table 4
Absolute and relative organ weight of F2 male and female weanlings.
AAS (ppm)		0 (control)	50	500	5000
Males
No. of animals		22	18	22	23
Body weight	(g)	89.9 ± 7.5	94.1 ± 8.3	91.7 ± 7.9	82.9 ± 10.2*
Brain	(g)	1.70 ± 0.06	1.73 ± 0.06	1.70 ± 0.06	1.67 ± 0.08
	(g/100 g b.w.)	1.90 ± 0.17	1.85 ± 0.14	1.86 ± 0.12	2.04 ± 0.23*
Thymus	(mg)	375 ± 69	379 ± 50	365 ± 50	296 ± 53**
	(mg/100 g b.w.)	417 ± 65	404 ± 53	399 ± 46	359 ± 58**
Liver	(g)	4.12 ± 0.55	4.49 ± 0.53	4.34 ± 0.44	3.69 ± 0.48*
	(g/100 g b.w.)	4.57 ± 0.32	4.77 ± 0.34	4.73 ± 0.19	4.46 ± 0.20
Kidney^a	(g)	1.02 ± 0.10	1.08 ± 0.10	1.03 ± 0.10	1.01 ± 0.13
	(g/100 g b.w.)	1.13 ± 0.08	1.15 ± 0.08	1.13 ± 0.07	1.22 ± 0.07**
Spleen	(mg)	390 ± 86	387 ± 48	393 ± 40	292 ± 52**
	(mg/100 g b.w.)	435 ± 93	413 ± 54	430 ± 41	352 ± 46**
Adrenal^a	(mg)	26.0 ± 3.8	25.2 ± 3.5	25.5 ± 3.3	24.7 ± 4.3
	(mg/100 g b.w.)	29.0 ± 4.1	26.7 ± 3.3	28.0 ± 3.9	29.8 ± 3.0
Testis^a	(mg)	546 ± 83	571 ± 83	572 ± 70	515 ± 67
	(mg/100 g b.w.)	607 ± 72	605 ± 59	623 ± 51	624 ± 65
Epididymis^a	(mg)	74.8 ± 7.5	76.1 ± 10.6	75.1 ± 9.9	68.7 ± 9.1
	(mg/100 g b.w.)	83.7 ± 10.2	80.7 ± 7.5	82.0 ± 9.0	83.6 ± 11.5
Females
No. of animals		22	18	22	23
Body weight	(g)	85.3 ± 7.2	87.6 ± 6.5	84.0 ± 4.9	77.2 ± 5.7**
Brain	(g)	1.65 ± 0.05	1.65 ± 0.06	1.64 ± 0.06	1.62 ± 0.06
	(% of body weight)	1.95 ± 0.16	1.89 ± 0.13	1.95 ± 0.08	2.11 ± 0.15**
Thymus	(mg)	367 ± 68	354 ± 60	352 ± 47	300 ± 40**
	(mg/100 g b.w.)	432 ± 84	405 ± 66	419 ± 48	390 ± 55
Liver	(g)	3.93 ± 0.41	3.97 ± 0.37	3.80 ± 0.33	3.33 ± 0.34**
	(g/100 g b.w.)	4.61 ± 0.25	4.54 ± 0.19	4.52 ± 0.25	4.31 ± 0.31**
Kidney^a	(g)	0.96 ± 0.08	0.97 ± 0.09	0.94 ± 0.06	0.93 ± 0.08
	(g/100 g b.w.)	1.13 ± 0.07	1.11 ± 0.07	1.12 ± 0.05	1.21 ± 0.07**
Spleen	(mg)	355 ± 53	330 ± 33	349 ± 52	276 ± 35**
	(mg/100 g b.w.)	416 ± 51	378 ± 35*	415 ± 59	358 ± 42**
Adrenal^a	(mg)	23.3 ± 2.3	23.2 ± 2.3	23.2 ± 3.4	23.2 ± 2.4
	(mg/100 g b.w.)	27.4 ± 2.7	26.6 ± 2.9	27.5 ± 3.5	30.0 ± 3.1*
Ovary^a	(mg)	24.6 ± 3.0	24.9 ± 4.0	24.2 ± 4.1	20.4 ± 3.2**
	(mg/100 g b.w.)	29.1 ± 4.3	28.5 ± 4.0	29.0 ± 5.5	26.7 ± 4.9
Uterus	(mg)	71.0 ± 55.7	66.8 ± 16.5	58.5 ± 11.8	53.5 ± 11.1*
	(mg/100 g b.w.)	82.3 ± 59.4	76.0 ± 15.4	69.6 ± 12.4	69.5 ± 14.8

Values are given as the mean ± S.D.

* Significantly different from the control, P < 0.05.

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

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

Conclusions:

In a two-generation study continuously exposing rats to aluminum ammonium sulfate via drinking water resulted in a NOAEL level of 500 ppm, which equals a dose of 5.35 mg Aluminum/kg bw/day. The LOAEL was 5000 ppm in this study.

Executive summary:

In a 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. Male and female rats of F0 and F1 received AAS from 10-weeks prior mating, mating and for the duration of female´s pregnancy. Males were sacrificed after parturition and females further received AAS during lactation. On PNDs 21-25 animals were selected as F1 parents marking day 0 of the 10-week prior mating phase.

Water consumption was decreased in all AAS-treated groups, and the body weight of parental animals transiently decreased in the 5000 ppm group. In either generation, no compound-related changes were found in estrous cyclicity, sperm parameters, copulation, fertility and gestation index, number of implantations and live birth pups, sex ratios of pups or viability during the preweaning period.

Male and female F1 pups in the 5000 ppm group showed a lower body weight on postnatal day 21, while there were no differences in the birth weight of F1 and F2 pups between the control and AAS-treated groups. Preweaning body weight gain in F2 males and females indicated a similar decreasing tendency at 5000 ppm. In F1 and F2 weanlings, the weight of the liver, spleen and thymus decreased at 5000 ppm, but no histopathological changes were found in these organs. In F1 females in the 5000 ppm group, vaginal opening was delayed slightly. There were no compound-related changes in male preputial separation or in other developmental landmarks. In behavioral tests conducted for F1 animals at 4–6 weeks of age, no compound-related changes were found in spontaneous locomotor activity and performance in a water-filled multiple T-maze. In conclusion, the NOAEL of AAS 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.

The two-generation study in the rat is classified acceptable and satisfies the guideline requirements for a development toxicity study (OECD 416) in rat.

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

Reference 2

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 report attached in section 13 of IUCLID.

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 justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

41 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

GLP-compliant studies rated as Klimisch 2 and conducted with read-across substances according to the relevant OECD TG 416 with acceptable restrictions.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

For human health endpoints, the relative bioavailability of different metal-ions from ALFERROCK would determine its potential to cause toxicological effects and also govern the severity of such effects. According to the results of the bioelution testing, aluminium has the highest relative release into artificial physiological media representing relevant exposure routes and therefore was considered as main constituent for the human health hazard/risk assessment of Alferrock (Klawonn, 2021, please see IUCLID section 7.1.1).

No data on reproductive toxicity is available for the test substance. However, suitable data is available from the read-across partners aluminium ammonium sulfate and aluminium sulfate. Due to higher water solubility of the read-across substance aluminium ammonium sulfate compared to the aluminium release from the test substance, the resulting bioavailability of aluminium from the test substance would be expected to be lower. The water solubility of the other read-across substance aluminium sulfate is in a similar range compared to the aluminium release from the test substance. Therefore, the read-across to the source substances aluminium potassium sulfate and aluminium sulfate is considered adequately protective. Details on the read-across rationale 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.

Effects on developmental toxicity

Description of key information

No developmental toxicity data is available for the target substance.

In the absence of substance specific data for Alferrock, a conservative approach to hazard evaluation is to assume that aluminium released from Alferrock shows the same systemic hazards as other aluminium compounds with similar or higher bioavailability. Read-across to aluminium compounds with a high release of aluminium ions (soluble Al salts) is considered to represent a worst-case approach that overestimates toxicological properties of the target compound. For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

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.

In a second developmental toxicity study (similar to OECD TG 414), aluminum hydroxide was orally administered to groups of 20 pregnant female Swiss mice/dose by gavage at dose levels of 0, 66.5, 133 and 266 mg/kg bw/day in distilled water from days 6 through 15 of gestation.

There were no treatment-related effects in maternal and developmental parameters observed. The maternal and developmental NOAEL is therefore considered to be >266 mg/kg bw/day (corresponding to 92.01 mg Al/kg bw/day).

Link to relevant study records

Reference

Reference 1

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 report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Description (incidence and severity):

No aluminum hydroxide-related clinical signs of toxicity were observed in the pregnant mice of any group.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No aluminum hydroxide-related deaths were observed in the pregnant mice of any group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Maternal toxicity was also assessed by examining the changes in maternal weight gain and food consumption. No significant differences between the aluminum hydroxide-treated groups and the control group were noted.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Upon sacrifice, the absolute and relative weights of heart, lungs, spleen, liver, kidneys and brain were similar between treated and control groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross postmortem effects attributed to the treatment were recorded.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The administration of aluminum hydroxide by oral gavage daily from day 6 to 15 of gestation had no recognizable adverse effects on the number of implantations and number of resorptions.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The administration of aluminum hydroxide by oral gavage daily from day 6 to 15 of gestation had no recognizable adverse effects on the number of resorptions.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The administration of aluminum hydroxide by oral gavage daily from day 6 to 15 of gestation had no recognizable adverse effects on the number of resorptions.

Dead fetuses:

no effects observed

Description (incidence and severity):

The administration of aluminum hydroxide by oral gavage daily from day 6 to 15 of gestation had no recognizable adverse effects on the number of live and dead fetuses.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not specified

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

> 266 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 92 mg/kg bw/day

Based on:

other: Aluminum content

Basis for effect level:

other: No adverse effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Body weights of fetuses removed on day 18 of gestation were not affected by any dose of aluminium hydroxide.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The administration of aluminum hydroxide by oral gavage daily from day 6 to 15 of gestation had no recognizable adverse effects on the number of live and dead fetuses.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The administration of aluminum hydroxide by oral gavage daily from day 6 to 15 of gestation had no recognizable adverse effects on the fetal sex ratio.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Body weights and body lengths of fetuses removed on day 18 of gestation were not affected by any dose of aluminium hydroxide.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No remarkable external malformations were found in any group.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No remarkable skeletal abnormalities were found in any group.

Visceral malformations:

no effects observed

Description (incidence and severity):

No remarkable internal soft-tissue defects were found in any group.

Key result

Dose descriptor:

NOAEL

Effect level:

> 266 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 92 mg/kg bw/day

Based on:

other: Aluminum content

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1: Maternal Relative Organ Weights of Mice Receiving Aluminum Hydroxide on Days 6 Through 15 of Gestation

	Dose (mg/kg/day)
	0		66.5		133		266
	mean	SE	mean	SE	mean	SE	mean	SE
Heart	0.31	0.01	0.35	0.03	0.34	0.03	0.35	0.02
Lungs	0.56	0.04	0.61	0.05	0.46	0.04	0.52	0.07
Spleen	0.23	0.02	0.25	0.03	0.20	0.01	0.27	0.04
Liver	4.39	0.16	4.82	0.17	4.25	0.27	4.26	0.28
Kidneys	0.81	0.03	0.89	0.07	0.88	0.06	0.89	0.09
Brain	0.79	0.03	0.77	0.09	0.87	0.07	0.87	0.10

Table 2: Effects of Aluminum Hydroxide Administered to Mice on Days 6 Through 15 of Gestation

	Dose (mg/kg/day)
	0		66.5		133		266
	mean	SE	mean	SE	mean	SE	mean	SE
No. of litters	20	-	18	-	19	-	18	-
No. of total implants/litter	11.50	0.70	12.40	0.70	11.80	0.70	11.10	1.00
No. of resorptions/litter
early	0.40	0.20	3.00	1.70	2.40	1.10	1.30	1.10
late	0.00	0.00	0.10	0.20	0.10	0.30	0.10	0.30
No. of live fetuses/litter	11.10	0.70	9.40	1.10	9.20	1.10	9.80	1.20
No. of dead fetuses/litter	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Sex ratio (M:F)/litter	1.34	0.25	0.96	0.07	1.02	0.17	1.13	0.25
Fetal body weight (g)	1.40	0.02	1.41	0.03	1.43	0.02	1.42	0.03
Fetal body length (cm)	3.11	0.02	3.11	0.03	3.24	0.03	3.10	0.03

Conclusions:

In a prenatal developmental toxicity study in Swiss mice, aluminum hydroxide did not induce maternal toxicity up to 266 mg/kg bw/day. Neither embryotoxicity nor teratogenicity was detected in any treatment group. The no observed adverse effect level (NOAEL) for maternal and developmental effects is therefore >266 mg/kg bw/day (corresponding to 92.01 mg Al/kg bw/day).

Executive summary:

In a developmental toxicity study (similar to OECD TG 414), aluminum hydroxide was orally administered to groups of 20 pregnant female Swiss mice/dose by gavage at dose levels of 0, 66.5, 133 and 266 mg/kg bw/day in distilled water from days 6 through 15 of gestation.

There were no treatment-related effects in mortality, clinical signs, body weight or maternal developmental parameters observed. The maternal NOAEL is therefore considered to be >266 mg/kg bw/day (corresponding to 92.01 mg Al/kg bw/day).

In addition, there were no treatment-related effects in developmental parameters in offspring. The developmental NOAEL is therefore considered to be >266 mg/kg bw/day (corresponding to 92.01 mg Al/kg bw/day).

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

Reference 2

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 report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Description (incidence and severity):

No maternal deaths or signs of toxicity were observed during the study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No maternal deaths or signs of toxicity were observed during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant maternal toxicity was observed at any Al(OH)3 dose level.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant maternal or developmental toxicity was observed at any Al(OH)3 dose level.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

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: No maternal deaths or signs of toxicity were observed during the study.

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

No embryotoxic/developmental 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: No embryotoxic/developmental effects were observed during the study.

Abnormalities:

no effects observed

Developmental effects observed:

no

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 justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

92 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Study design was comparable to the relevant OECD TG 414 with acceptable restrictions and conducted with read-across substances.

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

For human health endpoints, the relative bioavailability of different metal-ions from ALFERROCK would determine its potential to cause toxicological effects and also govern the severity of such effects. According to the results of the bioelution testing,aluminium has the highest relative release into artificial physiological media representing relevant exposure routes and therefore was considered as main constituent for the human health hazard/risk assessment of Alferrock (Klawonn, 2021, please see IUCLID section 7.1.1).

No data on developmental toxicity is available for the test substance. However, suitable data is available from the read-across partner aluminium hydroxide. Due to higher water solubility of the read-across substance aluminium hydroxide compared to the aluminium release from the test substance, the resulting bioavailability of aluminium from the test substance would be expected to be lower. Therefore, the read-across to the source substance aluminium hydroxide is considered adequately protective. Details on the read-across rationale are provided in section 13.

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.

In a second developmental toxicity study (similar to OECD TG 414), aluminum hydroxide was orally administered to groups of 20 pregnant female Swiss mice/dose by gavage at dose levels of 0, 66.5, 133 and 266 mg/kg bw/day in distilled water from days 6 through 15 of gestation.

There were no treatment-related effects in mortality, clinical signs, body weight or maternal developmental parameters observed. The maternal NOAEL is therefore considered to be >266 mg/kg bw/day (corresponding to 92.01 mg Al/kg bw/day).

In addition, there were no treatment-related effects in developmental parameters in offspring. The developmental NOAEL is therefore considered to be >266 mg/kg bw/day (corresponding to 92.01 mg Al/kg bw/day).

Justification for classification or non-classification

No adverse effects on reproduction and pre-natal development were reported in the selected studies. Based on the available data, classification of the test substance for reproductive toxicity according to the criteria of Annex I of Regulation (EC) 1272/2008 (CLP Regulation) is not warranted.