Fatty acids, C16-18 (even numbered), aluminum salts

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP-Guideline Study with acceptable restrictions (insufficient documentation in methods used, pH of the drinking water differed in control and test groups resulting in reduced water consumption, Al content was not analysed in blood, urine and tissues), tested with the source substance CAS 10043-01-3. In accordance to ECHA guidance document "Practical guide 6: How to report read-across and categories (May 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

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)

Deviations:

yes

Remarks:

insufficient documentation of methods used, the pH of the drinking water differed in control and test groups

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium sulphate

EC Number:

233-135-0

EC Name:

Aluminium sulphate

Cas Number:

10043-01-3

IUPAC Name:

aluminium sulphate

Details on test material:

- Name of test material (as cited in study report): Aluminium sulfate (AS), Al2(SO4)3
- Analytical purity: 98.5%
- Lot/batch No.: Lot No. 007x1828
- Stability in vehicle/water: stable in ion-exchanged water at concentrations of 0.1, 0.6 and 15 mg/mL for at least 10 days (storage for 4 days at room temperature following 6 days refrigerated storage).
- Stability under storage conditions: stored under cool and dark conditions in sealed containers
- pH: 3.57 - 4.20 (dosing solution)

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: (F0) 5 weeks
- Housing: animals were housed individually in suspended wire-mesh cages, except for the acclimation, mating and nursing periods.
From Day 17 of gestation to Postnatal Day 21, the wire-mesh floors were replaced with stainless-steel trays, and individual dams and litters were reared using wood chips as bedding (White Flake; Charles River Laboratories Japan, Inc., Yokohama, Japan).
- Diet: standard laboratory rodent chow (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan), ad libitum.
- Al content in diet: 25 - 29 ppm (analysed by atomic absorption spectrometry for each lot)
- Water: deionized drinking water, ad libitum
- Al content in water: < 5 µg Al/mL (drinking water of controls)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 – 25
- Humidity (%): 36 - 59
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

other: water (deionized)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared freshly at least every 6 days. Drinking solutions were replaced at least once every 4 days.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: for up to 2 weeks until successful mating
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): individually except for nursing period
- For F1 mating, cohabitation of siblings was avoided.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of aluminium sulphate in drinking water were analyzed using high performance liquid chromatography (the quantitation limit: 5 µg/mL) in the first and last preparations and once every 3 months (analysed Al concentrations: 97.5 – 106.3% of the target).

Duration of treatment / exposure:

(P) Males: 10 weeks before mating.
(P) Females: 10 weeks before mating, during mating and pregnancy, 26 days through weaning of their F1 offspring.
(F1) Males: 26 days at weaning, during growth into adulthood, mating and production of an F2 generation, until weaning of the F2 generation (postnatal day 26).
(F1) Females: 26 days at weaning, during growth into adulthood, mating and production of an F2 generation, until weaning of the F2 generation (postnatal day 26).

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

Dams (F0) were allowed to deliver spontaneously and nurse their pups. Litters were normalized on PND 4.
The day on which F1 parental animals were selected for F2 generation production, was designated as Day 0 of dosing for the F1 generation. Exposure of the F1 weanlings occurred at the same doses as their parents.
F1-selected rats were necropsied in the same manner as described for F0 rats. The remaining F1 weanlings and all F2 weanlings were necropsied on PND 26.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
120, 600 and 3000 ppm
Basis:
nominal in water

Doses / concentrations 2

Remarks:

Doses / Concentrations:
F0 males: 8.6, 41.0 and 188 mg Al2(SO4)3/kg bw/day; F0 females: 14.4, 71.5 and 316 mg Al2(SO4)3/kg bw/day
Basis:
other: mean dose value as calculated from the reported body weight and water consumption (including aluminium from diet and drinking water)

Doses / concentrations 3

Remarks:

Doses / Concentrations:
F0 males: 2.96, 8.06 and 31.2 mg Al/kg bw/day; F0 females: 4.5, 13.5 and 52.0 mg Al/kg bw/day
Basis:
other: total doses of aluminium (food and water combined)

Doses / concentrations 4

Remarks:

Doses / Concentrations:
F1 males: 10.7, 50.2 and 232 mg Al2(SO4)3/kg bw/day; F1 females: 15.3, 74.2 and 338 mg Al2(SO4)3/kg bw/day
Basis:
other: mean dose value as calculated from the reported body weight and water consumption (including aluminium from diet and drinking water)

Doses / concentrations 5

Remarks:

Doses / Concentrations:
F1 males: 3.55, 9.78 and 38.5 mg Al/kg bw/day; F1 females: 4.72, 14.0 and 55.6 mg Al/kg bw/day
Basis:
other: total doses of aluminium (food and water combined)

No. of animals per sex per dose:

24

Control animals:

other: yes, sham-exposed (total dose of aluminium via food and water: F0: 1.62 (males) and 2.29 (females) mg Al/kg bw/day; F1: 1.93 (males) and 2.35 (females) mg Al/kg bw/day)

Details on study design:

- Dose selection rationale: dose levels were based on the results of a foregoing range finding study, in which animals were orally exposed to 0, 1000, 3000, 10,000 and 30,000 ppm aluminium sulphate for 7 weeks.

Results: High-dose animals were euthanized after 2 weeks due to water avoidance. In the other groups, decreased water consumption was observed. Reduced food consumption and decreased body weights were observed at 3000, 10,000 and 30,000 ppm. Necropsy reevaled thickening of the limiting ridge in the stomach and atrophy of the thymus and spleen at 10,000 ppm. Relative weights of the liver, thymus and spleen were decreased in females given 3000 or 10,000 ppm. The body weights of pups on postnatal day (PND) 4 were decreased at 10,000 ppm. Based on these results, 120, 600 and 3000 ppm were selected as dose levels in the main study.

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: weekly. For dams, body weight was recorded on days 0, 7, 14 and 20 of gestation and days 0, 4, 7, 14 and 21 of lactation

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Time schedule for examinations: weekly. For dams, food consumption was recorded on days 0, 7, 14 and 20 of gestation and days 0, 7, 14 and 21 of lactation

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: twice a week, and on days 0, 4, 7, 11, 14, 17 and 20 of gestation and days 0, 4, 7, 11, 14, 17, 19 and 21 of lactation.

Oestrous cyclicity (parental animals):

Estrous cyclicity was tested throughout the last 2 weeks of the premating period and during cohabitation until evidence of copulation was detected via daily vaginal lavage.
Repeated 4 – 6 day estrous cycles were considered as normal.

Sperm parameters (parental animals):

Parameters examined in P/F1 male parental generations:
epididymis weight, sperm motility, sperm count in epididymides, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS: Yes
Performed on day 4 postpartum: maximum of 8 pups/litter (4/sex/litter); no adjustment was made for litters of fewer than eight pups.

PARAMETERS EXAMINED:
The following parameters were examined in F1 / F2 offspring: number of pups, sex of pups, live birth, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities. Clinical signs of toxicity (daily) and the body weight of live pups were measured on PND 0, 4, 7, 14 and 21.

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

DEVELOPMENTAL LANDMARKS
Developmental landmarks:
- Pinna unfolding in all F1 and F2 live pups (from PND 1 to PND 4).
- The anogenital distance (AGD) was measured using calipers on PND 4 in all F1 and F2 pups, and the normalized value of AGD to body weight, AGD/cube root of the body weight ratio, was calculated;
- Incisor eruption on one male and one female F1 and F2 pup selected from each litter were evaluated on PND 8. Eye opening was determined beginning on PND 12.
- The body weight of the respective F1 and F2 pups was recorded on the day the criteria were fulfilled.

Neuromotor performance:
- Surface righting reflex, negative geotaxis and mid-air righting reflex were assessed on PND 5, 8 and 18, respectively, for one male and one female F1 and F2 pup selected from each litter.

Sexual maturation:
- Preputial separation was recorded daily in all F1 males selected as F1 parents beginning on PND 35.
- Vaginal opening was recorded daily in all F1 females selected as F1 parents beginning on PND 25.
- The body weights of the respective F1 offspring were recorded on the day of completion of these pubertal landmarks.

NEUROBEHAVIOURAL EXAMINATIONS
Locomotor activity:
- Spontaneous locomotor activity was measured in 10 male and 10 female F1 rats which were randomly selected from each group at 4 weeks of age using a multi-channel activity monitoring system (SUPERMEX; Muromachi Kikai Co., Ltd., Tokyo, Japan).


T-maze test:
- A water-filled multiple T-maze test (Biel (1940) was conducted in 10 male and 10 female F1 rats selected randomly from each group at 6 weeks of age.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: as soon as possible after the last litters in each generation were produced (after parturition of their paired females).
- Maternal animals: after the last litter of each generation was weaned, on PND 26.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera;
- All female rats showing successful reproductive performance were evaluated for estrous cycle by examination of the vaginal smear after weaning.
- The number of uterine implantation sites was recorded for each dam.
- The testis and epididymis were prepared for microscopic examination and weighed.
- The brain, pituitary, thyroid, thymus, liver, kidneys, spleen, adrenals, testes, epididymides, seminal vesicles (with coagulating glands and their fluids), ventral prostate, uterus and ovaries in males and females were weighed before fixation and underwent macroscopic examination.
- The thyroid and seminal vesicles were weighed after fixation.
In 10 F1 females, randomly selected from the control and highest dose groups, the number of primordial follicles was counted in about 40 sections per ovary.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathological evaluations were performed:
- in all animals of the control and highest dose groups;
- in females with abnormal estrous cycles, abnormal delivery or totally dead pups;
- in males and females without evidence of copulation or insemination, and
- in all animals with grossly abnormal reproductive organs.
- testes and epididymis were fixed in Bouin’s solution and preserved in 70% ethanol; all other organs were fixed in 10% neutral buffered formalin.

Testes, epididymides, seminal vesicles, ventral prostate, coagulating gland, ovaries, uterus and vagina were sectioned, stained with hematoxylin–eosin and examined under a light microscope.
When the highest dose group showed treatment-related changes in some tissues, the same tissues from the next lower dose group were examined.

Postmortem examinations (offspring):

SACRIFICE
Following the adjustment of litter size on PND4, culled pups were euthanized and subjected to gross external and internal examinations. Pups found dead before weaning were necropsied immediately. Pups from the F1 offspring not selected as parental animals and all F2 pups were sacrificed on PND 26.

HISTOPATHOLOGY / ORGAN WEIGTHS
Organ weights were determined for 1 male/female F1 and F2 weanling selected from each dam:
- brain, thymus, liver, kidneys, spleen, adrenals, testes, epididymides, ventral prostate, uterus and ovaries
- since test substance-related organ weight changes were found in the liver and spleen of the highest dose group in F1 and F2 generations, these tissues were histopathologically examined from 10 male and 10 female F1 and F2 weanlings in the control and highest dose groups;
- if treatment-related histopathological changes were observed in the high-dose group, the same tissues from the next lower dose group were examined.

Statistics:

Homogeneity of distribution for parametric data (body weight, food and water consumption, length of the estrous cycle and gestation, precoital interval, the number of implantations and pups born, delivery index, reflex response time, age at sexual maturation, behavioural test parameters, organ weight and sperm parameters) a Bartlett’s test was performed.
Pre-weaning pups, body weight, AGD, viability, and age at the completion of developmental landmarks were similarly analysed using the litter as the experimental unit.
One Way Analysis of Variance was performed when the homogeneity of distribution was established.
If a significant difference was detected, Dunnett’s test was conducted for comparisons between control and individual treatment groups.
Data without homogeneity were analysed using the Kruskal–Wallis rank sum test. If significant differences were found, the Mann Whitney’s U test was conducted to compare control and each dosage group. Fisher’s exact test was used to compare the incidence of parental animals showing clinical signs, autopsy and histopathological findings, the incidence of females with normal estrous cycles, incidence of weanlings with histopathological findings, copulation, fertility and gestation index, neonatal sex ratio and completion rate of negative geotaxis between the AS and control group.
The Wilcoxon rank sum test was used to analyse the incidence of pups showing clinical signs and necropsy findings per litter, the completion rate of pinna unfolding in each litter, and the success rate of surface and mid-air righting reflex.
Student’s t-test was used to compare the number of primordial follicles in the control and highest dose group because the homogeneity of variance was indicated by the F-test.
All of these statistical analyses were conducted using the 5% level of probability as the criterion for significance.

Reproductive indices:

The following reproductive indices were calculated in F0 and F1 generation parental animals of each dose group:
- Copulation index (%) (no. of animals with successful copulation/no. of animals paired) ×100;
- Precoital interval (days);
- Fertility index (%) (no. of males that impregnated a female or no. pregnant/no. of animals with successful copulation) × 100;
- Gestation index (%) (no. of females that delivered live pups/no. of pregnant females) × 100;
- Gestation length (days);
- Delivery index (%) (no. of pups delivered/no. of implantations) × 100;
- Estrous cycle in F0 and F1 females

Offspring viability indices:

F1 and F2 offspring:
Maternal indices:
No. of litters;
No. of pups delivered;
Sex of all pups;
Sex ratio of pups total (no. of male pups/total no. of pups).

Viability index:
on PND 0 (%) = (no. of live pups on PND 0/no. of pups delivered) × 100;
on PND 4 (%) = (no. of live pups on PND 4/no. of live pups on PND 0) × 100;
on PND 21 (%) = (no. of live pups on PND 21/no. of live pups on PND 4 after cull) × 100.

Individual body weight:
Male and female individual weight during lactation on PND 0, 4, 7, 14 and 21.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

3000 ppm: significant reduction of body weight in F0 males/females in the first 2 -3 weeks of dosing; 600 and 3000 ppm: reduced food consumption in males and females

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

3000 ppm: significant reduction of body weight in F0 males/females in the first 2 -3 weeks of dosing; 600 and 3000 ppm: reduced food consumption in males and females

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: 120, 600 and 3000 ppm: significant decrease in water consumption in F0 males and females

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

persistent diestrus in in control and test animals (non-adverse)

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

3000 ppm: reduced number of cauda epdididymal sperm (non-adverse)

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No clinical signs of toxicty were observed in parental animals. 1 F0 female of the mid-dose group died at 2 weeks of gestation which showed a subcutaneous mass in the abdominal region starting 5 weeks of dosing.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
A sgnificant decrease in body weight was observed in high-dose males and females in the first 2 - 3 weeks of dosing. No direct correlation to reduced food consumption was present which was affected in mid- and high-dose animals to distinct time points:
F0 mid-dose males - during the first week of dosing, high-dose males: during weeks 1, 8 and 13-14;
F0 mid-dose females: during week 3 of lactation, high-dose females: 1 week of dosing and during week 3 of lactation.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
F0 and F1 females showed no significant alterations in the estrous cycle during the premating period. However, persistent diestrus was determined in a few control and test animals.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
High-dose males showed a significantly decreased absolute number of cauda epididymal sperm (253.8 ±61.3 × 106/cauda compared to 286.3 ±40.3 ×106/cauda in test and control animals, respectively). However, no difference was present after normalization to epididymal weight. Thus, the effect is considerd as non-adverse.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No significant differences on reproductive function or performance were observed between control and test animals including copulation (males, females), fertility (males, females), gestation index, the precoital interval, gestation length, delivery index, the number of implantations and number of litters or pups delivered.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Reduced organ weights of the liver (absolute and relative weight) and spleen (absolute weight) were found in high-dose males whereas no alterations were observed in females.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No dose-related gross lesions were found in animals of all dose groups.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No compound-related alterations were observed in high-dose animals. Therefore, organs from lower dose group animals were not examined in histopathology.

Effect levels (P0)

1

Dose descriptor:

other: LOAEL (systemic toxicity)

Effect level:

367.1 mg/kg bw/day (nominal)

Based on:

other: Fatty acids, C16-18(even numbered), aluminum salts

Sex:

male

Basis for effect level:

other: decreased body weight gain; decreased food consumption ; calculated value for Fatty acids, C16-18(even numbered), aluminum salts with a content of 8.5% Al (worst case assumption)

2

Dose descriptor:

other: LOAEL (systemic toxicity)

Effect level:

611.76 mg/kg bw/day (nominal)

Based on:

other: Fatty acids, C16-18(even numbered), aluminum salts

Sex:

female

Basis for effect level:

other: decreased body weight gain; decreased food consumption ; calculated value for Fatty acids, C16-18(even numbered), aluminum salts with a content of 8.5% Al (worst case assumption)

3

Dose descriptor:

other: NOAEL (sytemic toxicity)

Effect level:

94.82 mg/kg bw/day (nominal)

Based on:

other: Fatty acids, C16-18(even numbered), aluminum salts

Sex:

male

Basis for effect level:

other: calculated value for Fatty acids, C16-18(even numbered), aluminum salts with a content of 8.5% Al (worst case assumption)

4

Dose descriptor:

other: NOAEL (systemic toxicity)

Effect level:

158.8 mg/kg bw/day (nominal)

Based on:

other: Fatty acids, C16-18(even numbered), aluminum salts

Sex:

female

Basis for effect level:

other: calculated value for Fatty acids, C16-18(even numbered), aluminum salts with a content of 8.5% Al (worst case assumption)

5

Dose descriptor:

other: NOAEL (fertility)

Effect level:

367.1 mg/kg bw/day (nominal)

Based on:

other: Fatty acids, C16-18(even numbered), aluminum salts

Sex:

male

Basis for effect level:

other: calculated value for Fatty acids, C16-18(even numbered), aluminum salts with a content of 8.5% Al (worst case assumption)

6

Dose descriptor:

other: NOAEL (fertility)

Effect level:

611.76 mg/kg bw/day (nominal)

Based on:

other: Fatty acids, C16-18(even numbered), aluminum salts

Sex:

female

Basis for effect level:

other: calculated value for Fatty acids, C16-18(even numbered), aluminum salts with a content of 8.5% Al (worst case assumption)

7

Dose descriptor:

other: LOAEL (developmental toxicity)

Effect level:

654.12 mg/kg bw/day (nominal)

Based on:

other: Fatty acids, C16-18(even numbered), aluminum salts

Sex:

female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: F1 and F2 (migrated information)

8

Dose descriptor:

other: LOAEL (developmental toxicity)

Effect level:

452.94 mg/kg bw/day (nominal)

Based on:

other: Fatty acids, C16-18(even numbered), aluminum salts

Sex:

male

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: F1 and F2 (migrated information)

9

Dose descriptor:

other: NOAEL (developmental toxicity)

Effect level:

115.1 mg/kg bw/day (nominal)

Based on:

other: Fatty acids, C16-18(even numbered), aluminum salts

Sex:

male

Basis for effect level:

other: calculated value for Fatty acids, C16-18(even numbered), aluminum salts with a content of 8.5% Al (worst case assumption)

Remarks on result:

other: Generation: F1 and F2 (migrated information)

10

Dose descriptor:

other: NOAEL (developmental toxicity)

Effect level:

164.7 mg/kg bw/day (nominal)

Based on:

other: Fatty acids, C16-18(even numbered), aluminum salts

Sex:

female

Basis for effect level:

other: calculated value for Fatty acids, C16-18(even numbered), aluminum salts with a content of 8.5% Al (worst case assumption)

Remarks on result:

other: Generation: F1 and F2 (migrated information)

11

Dose descriptor:

other: LOAEL (systemic toxicity)

Effect level:

3 000 ppm (nominal)

Based on:

test mat.

Remarks:

equivalent to 31.2 (males) and 52.0 (females) mg Al/kg bw/day

Sex:

male/female

Basis for effect level:

other: decreased body weight gain; decreased food consumption

12

Dose descriptor:

other: NOAEL (systemic toxicity)

Effect level:

600 ppm (nominal)

Based on:

test mat.

Remarks:

(equivalent to 8.06 (males) and 13.5 (females) mg Al/kg bw/day)

Sex:

male/female

13

Dose descriptor:

other: NOAEL (fertility)

Effect level:

3 000 ppm (nominal)

Based on:

test mat.

Remarks:

equivalent to 31.2 (males) and 52.0 (females) mg Al/kg bw/day, P generation; equivalent to 38.5 (males) and 55.6 (females) mg Al/kg bw/day, F1 generation)

Sex:

male/female

Remarks on result:

other: Generation: P and F1 (migrated information)

14

Dose descriptor:

other: LOAEL (developmental toxicity)

Effect level:

3 000 ppm (nominal)

Based on:

test mat.

Remarks:

equivalent to 38.5 (males) and 55.6 (females) mg Al/kg bw/day

Sex:

male/female

Basis for effect level:

other: decreased preweaning body weight gain in the F1 and F2 males and females; decreased absolute and relative liver and spleen weights in the F1 and F2 males and females; delayed vaginal opening in the F1 females

Remarks on result:

other: Generation: F1 and F2 (migrated information)

15

Dose descriptor:

other: NOAEL (developmental toxicity)

Effect level:

600 ppm (nominal)

Based on:

test mat.

Remarks:

equivalent to 9.78 (males) and 14.0 (females) mg Al/kg bw/day

Sex:

male/female

Remarks on result:

other: Generation: F1 and F2 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F1 generation: 1 control pup showed trauma in the perianal region and tail, 1 low-dose pup had hemimelia and oligodactyly (non-adverse)

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1 generation: reduced body weight in high-dose males/females on PND 21 and at scheduled sacrifice; F2 generation: reduced body weight in high-dose females on PND 21 and at sacrifice in both genders

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

F1 females: delayed vaginal opening in high-dose females

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

F1 and F2 generation: alterations in liver, spleen, thymus, kidney, testes, epididymides, uterus, ovary and brain weights

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
Viability indeces of PND 0, 4 and 21 were comparable amomg the groups in either generation (F1 and F2).

CLINICAL SIGNS (OFFSPRING)
F1 generation
No significant difference between control and treated groups were determined. Findings of a trauma in the perianal region and tail or hemimelia and oligodactyly were found incidentally in 1 control and 1 low-dose pup, respctively.

F2 generation
No effects were observed in any dose group.

BODY WEIGHT (OFFSPRING)
F1 generation
Body weights of high-dose males and females were significantly lower on PND 21 and at sacrifice compared to the control.
F2 generation
Reduced body weight was determined in high-dose females on PND 21 and at sacrifice in both genders.

For details, please refer to Tables 1 and 2.


SEXUAL MATURATION (OFFSPRING)
F1 generation
Vaginal opening was significantly delayed in high-dose females (31.4 ± 1.7 compared to 29.5 ± 2.1 days in control) which went along with a slightly higher body weight compared to controls (119.0 ± 13.3 versus 109.6 ± 11.6 g).
Otherwise, no significant differences were noted regarding the age of preputial separation and no changes in body weights at the time of preputial completion were noted.


ORGAN WEIGHTS (OFFSPRING)
F1 generation
Significant decreases in liver (absolute and relative organ weight), spleen, thymus, kidney, testes and epididymides weights (absolute weights) and increased relative brain weights were observed in high-dose animals whereas the absolute uterus weight was decreased.

F2 generation
Weights of the thymus, spleen (absoulte and relative weights), liver and epididymides (absolute) were significantly reduced whereas relative brain weight was increased in high-dose males. Alterations in females were only noted in the mid-dose group, where reduced absoulte brain weight was obsreved.

For details, please refer to Tables 1 and 2.


GROSS PATHOLOGY (OFFSPRING)
No treatment-related alterations were found in either the F1 or F2 generation during the preweaning period (data not shown).


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

OTHER FINDINGS (OFFSPRING)
SEX RATIO
The sex ratio of pups was comparable among all test groups in either the F1 or F2 generation.

PHYSICAL DEVELOPMENT
The completion rate of pinna unfolding, and the age at completion of incisor eruption and eye opening was comparable among the groups in the F1 generation and F2 males of all test groups. In F2 females, completion rate of pinna unfolding on PND 2 was significantly lower compared to controls in the mid-dose group (17.0 ± 35.4%, compared with 45.8 ± 46.9 in mid-dose females and controls, respectively). Otherwise, females of the F2 generation did not show significant differences in regard to the measured developmental landmarks.

AGD and AGD per cube root of body weight ratio were comparable among the test groups in either the F1 and F2 generation.

NEUROMOTOR DEVELOPMENT
No significant changes were observed in the development of reflexes (surface righting reflex on PND5, negative geotaxis reflex on PND8 and midair righting reflex on PND 18) in any dose group of the F1 or F2 generation. Futher, the response times of surface righting and negative geotaxis reflexes were not affected by the treatment with the test substance. However, 1 mid-dose F2 female did not achieve the mid-air righting reflex on PND 18 in one of 3 trials without affecting the mean success rate between the control and mid-dose group (100 ± 0.0% versus 98.4 ± 7.3%).

RESULTS ON PARENTAL ANIMALS OF THE F1 GENERATION
CLINICAL SIGNS AND MORTALITY
No clinical signs of toxicity were observed in the parents received from the F1 generation. Incidental mortalities were observed in the low- and high-dose group: 1 low-dose male died at 9 weeks of dosing. Prior to death, soiling of periocular and perinasal fur and decreased locomotor activity were observed. Necropsy revealed ascitic, accumulation of pleural fluid and dark purple discoloration of the liver and kidneys. Further, 1 high-dose male died at week 12 of dosing without showing clinical signs of toxicity.

WATER CONSUMPTION
Statistically decreased water consumption was observed in the F1 generation in all dose groups: Low-dose males showed significantly decreased water consumption during 3-6, 8, 10 weeks of dosing whereas water consumption of mid-and high-dose animals was reduced throughout the dosing period. F1 females of the low-dose group showed reduced water consumption during 9 - 10 week of dosing, mid-dose females during week 10 of dosing and week 3 of lactation whereas high-dose females showed reduced water consumption throughout the dosing period.

FOOD CONSUMPTION
Significantly decreased food consumption was observed in the mid- and high-dose group either in week 10 of dosing (males) or week 3 of lactation (females). Moreover, low-dose females showed reduced food consumption during week 6 of dosing.

BODY WEIGHT
Body weights were comparable among the groups except for F1 low-dose females which revealed an increased body weight during week 6 - 8 compared to controls.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No significant alterations were determined in the estrous cycle of F1 females although some control and test animals had persistent diestrus.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
In contrast to F0 parental animals, no effect on the number of cauda epididymal sperm was determined in F1 males. Further, 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 was unaffected in F1 males.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproductive performance of F1 parental animals was not altered by the treatment. Copulation (males, females), fertility (males, females), gestation index, the precoital interval, gestation length, delivery index, the number of implantations, number of litters or pups delivered were comparable among the groups.

ORGAN WEIGHTS
In adult F1 males of the high-dose group, absolute weight of adrenals was significantly decreased compared to control animals. Further, mid-dose males showed significantly reduced absolut testis weight.
In contrast, females of the F1 generation did not differ from controls in respect to organ weights and the number of primordial follicles in the ovary was unaffected.

PATHOLOGY
No dose-related gross lesions were observed in the F1 generation. Further, reproductive organs revealed no compound-related alterations in adults of the F1 generation.

Effect levels (F1)

1

Dose descriptor:

other: NOAEL (fertility)

Generation:

F1

Effect level:

452.94 mg/kg bw/day (nominal)

Based on:

other: Fatty acids, C16-18(even numbered), aluminum salts

Sex:

male

Basis for effect level:

other: calculated value for Fatty acids, C16-18(even numbered), aluminum salts with a content of 8.5% Al (worst case assumption)

2

Dose descriptor:

other: NOAEL (fertility)

Generation:

F1

Effect level:

654.12 mg/kg bw/day (nominal)

Based on:

other: Fatty acids, C16-18(even numbered), aluminum salts

Sex:

female

Basis for effect level:

other: calculated value for Fatty acids, C16-18(even numbered), aluminum salts with a content of 8.5% Al (worst case assumption)

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1.Organ weights of F1 and F2 male weanlings

 

	Control		120 ppm		600 ppm		3000 ppm
	F1 males	F2 males	F1 males	F2 males	F1 males	F2 males	F1 males	F2 males
Number of animals	22	21	20	18	22	22	22	21
Body weight (%)	100%	100%	NS	NS	NS		87.44**	90.31**
Brain
Absolute weight (%)	100%	100%	NS	NS	NS	NS	NS	NS
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	113.22**	112.11**
Thymus
Absolute weight (%)	100%	100%	NS		NS	NS	81.33**	79.84**
Relative weight (%) (g/100 g bw)	100%	100%	NS	89.29*	NS	NS	NS	87.92**
Liver
Absolute weight (%)	100%	100%	NS	NS	NS	NS	80.60**	87.78**
Relative weight (%)(g/100 g bw)	100%	100%	NS	NS	NS	NS	91.61**	NS
Kidney^a
Absolute weight (%)	100%	100%	NS	NS	NS	NS	89.62**	NS
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	NS	NS
Spleen
Absolute weight (%)	100%	100%	NS	NS	NS	NS	76.40**	80.43**
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	86.93**	88.36**
Testis^a
Absolute weight (%)	100%	100%	NS	NS	NS	NS	90.44*	NS
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	NS	NS
Epididymis^a
Absolute weight (%)	100%	100%	NS	NS	NS	NS	88.02**	93.62*
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	NS	NS

NS: no statistically significant differences were observed

**: significantly different from the control, p<0.05;

*: significantly different from the control, p<0.01;

^a: value represents the total weights of the organs on both sides.

 

Table 2. Organ weights of F1 and F2 female weanlings

 

	Control		120		600		3000
	F1 females	F2 females	F1 females	F2 females	F1 females	F2 females	F1 females	F2 females
Number of animals	22	22	20	18	22	21	21	21
Body weight (%)	100%	100%	NS		NS		89.91**	91.34**
Brain
Absolute weight (%)	100%	100%	NS	NS	NS	102.5*	NS	NS
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS		110.20**	110.05**
Thymus
Absolute weight (%)	100%	100%	NS	NS	NS	NS	81.72**	NS
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	NS	NS
Liver
Absolute weight (%)	100%	100%	NS	NS	NS	NS	84.80**	86.24**
Relative weight %) (g/100 g bw)	100%	100%	NS	NS	NS	NS	94.26*	94.56**
Kidney^a
Absolute weight (%)	100%	100%	NS	NS	NS	NS	NS	NS
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	NS	NS
Spleen
Absolute weight (%)	100%	100%	NS	NS	NS	NS	86.65*	84.11**
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	NS	NS
Ovary^a
Absolute weight (%)	100%	100%	NS	NS	NS	NS	NS	84.52**
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	NS	NS
Uterus
Absolute weight (%)	100%	100%	NS	NS	83.85*	NS	78.47**	81.49*
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	NS	NS

NS: no statistically significant differences were observed

**: significantly different from the control, p<0.05;

*: significantly different from the control, p<0.01;

^a: value represents the total weights of the organs on both sides.