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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2008-2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Remarks:
The publication describes a study performed under GLP at the Safety Research Institute Kanto Chemical Compounds Co. Ltd, Sapporo Japan
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Aluminium sulphate
EC Number:
233-135-0
EC Name:
Aluminium sulphate
Cas Number:
10043-01-3
Molecular formula:
Al.3/2H2O4S
IUPAC Name:
aluminium sulphate
Details on test material:
Not reported
Specific details on test material used for the study:
lot: 007X1828
purity: 98.5%
storange of the sample: cool and dark in sealed container

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Atsugu Breeding Center, Charles River Laboratories Japan Inc. Yokohama, Japan
- Females: no data
- Age at study initiation: (P) 5 weeks
- Weight at study initiation: (P) Males: ca 180 g; Females: ca 120 g; (F1) Males: 80 g; Females: 70 g Values taken from figures in the publication)
- Fasting period before study: no
- Housing:
- Diet standard rat diet (CRF-1 Oriental Yeast Co. Ltd, Tokyo, Japan containing 25-29 ug/g Al) ad libitum
- Water: ionexchanged water containing the substance ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25 °C
- Humidity (%): 35-59 %
- Air changes (per hr): 10-15/h
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: drinking water
Details on exposure:
WATER PREPARATION
- Rate of preparation of diet (frequency): every 6 days
- Storage temperature of water: cooled
- FRequency based on stability for 4 days at room temperature and 6 days refrigerated (tested at 0.1, 0.6 and 15 mg/mL)

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 2 weeks
- Further matings after two unsuccessful attempts: not indicated
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In first and last preparation and every three months by HPLC (97.5-106.3% of nominal)
Drinking water was below the LOQ (0.5 ug/mL)
Duration of treatment / exposure:
P: 10 weeks and during mating gestation and lactation (parental males were necropsied at the same time as females)
F1: after weaning until mating and during mating gestation and lactation
Necropsy for non-selected F1 and F2 pups on day 26 after weaning
Frequency of treatment:
continuously
Details on study schedule:
- F1 parental animals not mated until unknown] weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21-25 days of age.
Doses / concentrationsopen allclose all
Dose / conc.:
0.12 mg/L drinking water
Dose / conc.:
0.6 mg/L drinking water
Dose / conc.:
3 mg/L drinking water
No. of animals per sex per dose:
24 males and 24 females for mating in P and F1 generation
Control animals:
other: ion exchanged drinking water
Details on study design:
- Dose selection rationale: based on a pre-test during 6-8 weeks, mating gestation and until day 4 post partum (at 1, 3, 10 and 30 mg/L in drinking water, NOAEL 3 mg/L)

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (females during gestation on day 0, 7, 14 and 20; during lactation on day 0, 7, 14 and 21)

FOOD CONSUMPTION : Yes
- Time schedule for examinations:weekly (females during gestation on day 0, 7, 14 and 20; during lactation on day 0, 7, 14 and 21)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: twice weekly females during gestation on day 0,4, 7, 11, 14, 17 and 20; during lactation on day 0, 4, 7, 11, 14, 17, 19 and 21)
- Calculation intake: based on mean values for body weight and water intake per group


OTHER:
Oestrous cyclicity (parental animals):
2 weeks pre-mating and during cohabitation (4-6 days oestrus cycle considered as normal)
Sperm parameters (parental animals):
Parameters examined in all adult male parental generations:
testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes to 4 males and 4 females
excess pups were killed and necropsied (gross external and internal observations)

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, body weight, physical or behavioural abnormalities, pinna unfolding, anogenital distance (AGD)(
In one male + one female pup per litter:onset and completion of incisor eruption, eye opening, perpetual separation and vaginal opening

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

ASSESSMENT OF BEHAVIOURAL EFFECTS:
On day 5, 8 and 18 in one male + one female pup per litter: surface rightening reflex, negative geitaxis, mid-air rightening reflex
After 4 weeks in 10/sex/group: motoractivity and swim test (T-maze)
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at the same time as the females]
- Maternal animals: All surviving animals depending on oestrus cycle status after weaning

GROSS NECROPSY
- Gross necropsy in all animals consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera; females counts of implementation sites; males testis and epididymides preservation:

HISTOPATHOLOGY / ORGAN WEIGHTS:
From all animals: weights of brain, pituitary, thyroids, thymus, liver, kidneys, spleen, adrenals, testes, epididymides,, seminal vessels, prostate, uterus, ovaries
From control and high dose (+ all females with abnormalities + non copulating animals + animals with abnormalities ate the sex organs): histopathology of testes, epididymides,, seminal vessels, coagulating gland, ventral prostate, uterus, ovaries and vagina
From 10 females from control and high dose group: count of premordial follicles in the right ovary
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 26 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
Organ weight of for 1 male + 1 female F1/F2 weanling/litter: brain, thymus, liver, kidneys, spleen, adrenals, testes, epididymides,, seminal vessels, prostate, uterus, ovaries
Microscopic examination of 10 males + females in control and high dose group of spleen and liver
Statistics:
Bartlett's test, Dunnett's test, Kruskal-Wallis test, Mann-Whitney's U-test, Fisher exact test, Student t-test (f-test)
Reproductive indices:
Copulation Index; Fertility Index; Gestation Index; Delivery Index
Offspring viability indices:
Viability Index on post-natal day 0, 4 and 21

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
P0: 1 female at 0.6 mg/L (mass)
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Decreased body weight at 3 mg/L during week 2 and 3 in males = females
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males: sign decreased at 0.6 and 3 mg/L during week 1; sign decreased during week 8 and 13-14 at 3 mg/L
Females: sign decreased at 3 mg/L during week 1; sign decreased during week 3 of lactation at 0.6 and 3 mg/L
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
significantly decreased in all dose groups (related to the acidity of the formulated water)
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related effects

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No treatment related effects
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
at 3 mg/L sign decrease in cauda epidydimal sperm (control 268E06/cauda; treated 254E06/cauda)
Reproductive performance:
no effects observed
Description (incidence and severity):
Copulation Index (%)
Males: 91.7, 91.7, 100. 91.7 at 0, 0.12,0.6 and 3 mg/L
Females:95.8, 100, 100, 100 at 0, 0.12,0.6 and 3 mg/L
Fertility Index (%):
Males: 95.5, 90,9. 100, 95.5 at 0, 0.12,0.6 and 3 mg/L
Females: 95.7, 91.7, 100, 91.8 at 0, 0.12,0.6 and 3 mg/L

Gestation Index (%): 100, 95.5, 95.7, 95.7 at 0, 0.12,0.6 and 3 mg/L
Delivery Index (%): 94.3, 88.6, 90.7, 92.0 at 0, 0.12,0.6 and 3 mg/L

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
13.5 mg/kg bw/day (actual dose received)
Based on:
element
Sex:
male
Basis for effect level:
food consumption and compound intake
water consumption and compound intake
organ weights and organ / body weight ratios
reproductive function (sperm measures)
Dose descriptor:
NOAEL
Effect level:
8.06 mg/kg bw/day (actual dose received)
Based on:
element
Sex:
female
Basis for effect level:
food consumption and compound intake
water consumption and compound intake
Remarks on result:
other: related to the acidity of the drinking water

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
31.2 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
cauda epididymis
Treatment related:
no
Dose response relationship:
no
Relevant for humans:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
F1: 1 male at 0.12 mg/L and 1 male at 3 mg/L
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant difference with controls
Females at 3 mg/L showed increased body weight during week 6-8
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males: sign decreased during week 10 at 0.6 and 3 mg/L
Females: sign decreased during week 3 of lactation at 0. and 3 mg/L
Incidental increases were also reported
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Males: sign decreased at 0.6 and 3 mg/L; sign decreased during week 3-6, 8 and 10 at 0.12 mg/L
Females: sign decreased at 3 mg/L; sign decreased during week 10 of dosing and 3 of lactation at 0.6 mg/L; sign decreased during week 9-10 at 0.12 mg/L
Effects related to the acidity of the formulations
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No difference with control animals
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No difference with control animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related effects
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related effects

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No treatment related effects
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No treatment related effects
Reproductive performance:
no effects observed
Description (incidence and severity):
Copulation Index (%)
Males: 95.8. 91.3, 95.8, 87.5 at 0, 0.12,0.6 and 3 mg/L
Females:100, 95.8, 100, 95.8 at 0, 0.12,0.6 and 3 mg/L
Fertility Index (%):
Males:91.3, 81.0, 91.3, 95.2 at 0, 0.12,0.6 and 3 mg/L
Females: 91.7, 82.6, 91.7, 91.3 at 0, 0.12,0.6 and 3 mg/L

Gestation Index (%): 100, 94.7, 100, 100 at 0, 0.12,0.6 and 3 mg/L
Delivery Index (%): 94.0, 87.5, 91.4, 94.6 at 0, 0.12,0.6 and 3 mg/L

Effect levels (P1)

Dose descriptor:
NOAEL
Effect level:
>= 9.78 - <= 14 mg/kg bw/day (actual dose received)
Based on:
element
Sex:
male/female
Basis for effect level:
food consumption and compound intake
water consumption and compound intake
Remarks on result:
other: No treatment related effects

Target system / organ toxicity (P1)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
see P1
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
see P1
Viability Index:
post natal day 0 (%): 100, 99.3, 99.7 and 99,5 at 0, 0.12, 0.6 and 3 mg/L
post natal day 4 (%): 98.7, 95.2, 98.8 and 98.0 at 0, 0.12, 0.6 and 3 mg/L
postnatal day 21 (%): 99.4, 100, 100 and 99.4 at 0, 0.12, 0.6 and 3 mg/L
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see P1
post natal day 21: at 3 mg/L sign decreased body weight in both sexes
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see P1
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
see P1
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
see P1
sign decreased abs thymus and spleen weight in both sexes at 3 mg/L
sign decreased abs kidney, testes and epididymus weight in males at 3 mg/L
sign decreased abs uterus weight in females at 0.6 and 3 mg/L
sign increased rel brain weight in both sexes at 3 mg/L
Gross pathological findings:
no effects observed
Description (incidence and severity):
see P1
No treatment related effects
Histopathological findings:
no effects observed
Description (incidence and severity):
see P1
No treatment elated effects in liver and spleen
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Delayed vaginal openeing in females at 3 mg/L
No other treatment related developmental effects

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment related behavioural effects (no dose response)

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
13.5 mg/kg bw/day (actual dose received)
Based on:
element
Sex:
female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
other: developmental toxicity (vaginal opening delayed)
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
8.06 mg/kg bw/day (actual dose received)
Based on:
element
Sex:
male
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios

Results: F2 generation

General toxicity (F2)

Clinical signs:
not specified
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Viability Index:
post natal day 0 (%): 99.7, 99.6, 99.4 and 99.7 at 0, 0.12, 0.6 and 3 mg/L
post natal day 4 (%): 94.7, 98.1, 99.1and 99.0 at 0, 0.12, 0.6 and 3 mg/L
postnatal day 21 (%): 100, 98.6, 100 and 100 at 0, 0.12, 0.6 and 3 mg/L
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
post natal day 21: at 3 mg/L sign decreased body weight in females
Food consumption and compound intake (if feeding study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
sign decreased abs spleen, abs thymus and rel brain weight in males at 3 mg/L
sign decreased abs liver and epididymus weight in males at 3 mg/L
sign decreased abs spleen, ovary and uterus weight in females at 3 mg/L
sign decreased abs/rel liver weight in females at 3 mg/L
sign increased rel brain weight in females at 3 mg/L
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related effects
Histopathological findings:
no effects observed
Description (incidence and severity):
No treatment related effects in liver and spleen
Other effects:
no effects observed
Description (incidence and severity):
No treatment related developmental effects

Developmental neurotoxicity (F2)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment related behavioural effects

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 9.78 - <= 14 mg/kg bw/day (actual dose received)
Based on:
element
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
31.2 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
In parental animals the most prominent effect of the substance was the decreased water intake (most likely due to the acidity of the formulation). THis has lead to effects on food intake and body weight at 3 mg/L. The NOAEL for parental toxicity is therefore set at 8.06 mg/kg bw in a worst case approch
Effects on sperm parameters were seen in parental males (P0) at 3 mg/L. No effects on reproduction were seen in the P0 and P1 generation. The NOAEL for reproduction toxicity is set at 8.06 mg/kg bw in a worst case approch
In the offspring (F1 and F2) effects were limited to a lowered body weight and some changes in organ weights at 3 mg/L. Delayed valginal opening was observed in F1 females at 3 mg/L. The NOAEL for developmental effects is therefore set at 8.06 mg/kg bw.
Executive summary:

In a two-generation reproductive toxicity study, male and female rats were given aluminium sulfate (AS) in drinking water at 0, 120, 600 or 3000 ppm. AS reduced water consumption in all treatment groups, and body weight was transiently decreased in the 3000 ppm group. In the F1 and F2 pups, preweaning body weight gain was inhibited at 3000 ppm, and the liver and spleen weight was 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. The data indicated that the NOAEL of AS in this two-generation study is 600 ppm for parental systemic toxicity and reproductive/developmental toxicity. The total ingested dose of aluminium from drinking water and food (standard rat diet, containing 25-29 ppm of aluminium) combined for this 600 ppm group was calculated to be 8.06 mg Al/kg bw/day.