Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From November 25th, 1998 to January 21st, 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: See read-across justification in section 13. Reliability of the original study 1.
Justification for type of information:
See attached document in section 13.
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From November 25th, 1998 to January 21st, 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: See read-across justification in section 13. Reliability of the original study 1.
Justification for type of information:
See attached document in section 13.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 27th, 1995
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl: CD®BR
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Age at study initiation: 68 to 72 days (age at start of adaptation)
- Weight at study initiation: males: 264 - 287 g; females: 195 - 216 g
- Housing: singly in MAKROLON cages type lll (except during the mating period). Granulated textured wood was used as bedding material in the cages. The cages were cleaned and changed once a week. Periodic analysis of the bedding material is conducted at least once a year. For identification, each rat received a continuous number between 1 and 80: a pattern of points was set on paws and/or tail by tattoo, additionally, the animal cages were labelled with the tattooed serial number, sex, study code number, type of study, route of administration, dose level, date of conception and dates of administration.
- Diet: ALTROMIN 1314 ad libitum. Samples of the food are analysed for contaminants twice a year.
- Water: tap water ad libitum. Samples of the drinking water are taken periodically and periodic analyses are performed.
- Acclimation period: 5 acclimatisation days prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2 °C
- Humidity (%): relative humidity 50% ± 15%
- Photoperiod (hrs dark / hrs light): the rooms were alternately lit (about 150 lux at approximately 1.5 m room height) and darkened in a 12 hours dark/12 hours light cycle.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: the test substance was suspended in tap water and was administered orally at a constant volume of 10 ml/kg bw 7 days per week during all duration of treatment (different for males and females). The control animals received 10 ml tap water/kg bw/day in the same manner. The test substance preparations were freshly prepared every day. The daily dose of the test substance was adjusted to the animal's body weight daily.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The amount of test item in aqueous samples obtained during the study was determined applying gas chromatography with flame ionization detection (GC/FID). The samples were analysed after addition of an internal standard and liquid/liquid extraction with ethyl acetate. The extracts were diluted and analysed by GC/FID. 18 samples from the aqueous application solutions were withdrawn at the start of the application phase (test day 1) for the determination of the stability, the homogeneity and the test substance concentration. Further, 3 samples were taken from the application solutions at the end of the application phase (test day 40) for the determination of the test substance concentration (total of 21 samples).

CHROMATOGRAPHY CONDITION:
- Column: HP-1 (100% methyl silicone), 25 m, 0.32 mm i.d., 0.52 /Jm film (Hewlett-Packard)
- Injector: 220 °C
- Detector: FID, 280 °C
- Oven: hold 50 °C (2 min.), heat up to 150 °C (20 °C/min.), hold 150 °C (2 min.)
- Carrier gas: Helium, 2.5 ml/min
- Split: 51 ml/min.
- Septum purge: 2.6 ml/min.
- Retention times: test item: approx. 4.7 min. Internal standard: approx. 3.9 min

PRECISION: coefficient of variation of 1.54 % (peak area ratios) indicated a stable and reliable GC system.

LINEARITY: r2= 0.998 : the method proved to be linear over the entire measuring range.

ACCURACY AND PRECISION: Accuracy and precision were within 1-10% (% of nominal concentration or coefficient of variation of the results of repeated analyses).

LOD = 0.654 mg/ml

CONCLUSION: the actual concentrations of the samples were within the range of 91.8 - 110.0 % of the nominal test item concentrations indicating correctly prepared, homogenous and stable mixtures.
Details on mating procedure:
- Impregnation procedure: cohoused.
- M/F ratio per cage: 1 male and 1 female animal were placed per cage (monogamous mating)
- Length of cohabitation: maximum 2 weeks.
- After 2 weeks of unsuccessful pairing replacement of first male by another male with proven fertility. This procedure was repeated until at least 8 pregnant dams were available for each group.
- Proof of pregnancy: each morning the females were examined for the presence of sperm. The day of conception (day 0 of pregnancy) was considered to be the day on which sperm was found.
Duration of treatment / exposure:
Males: 2 weeks prior to mating, during the mating period and approx. 2 weeks post mating at least until the minimum total dosing period of 28 days had been completed (up to and including the day before sacrifice).
Females: throughout the study beginning 2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.
Frequency of treatment:
Daily 7 days per week for all the duration of treatment (different for males and females).
Duration of test:
- Start of the study (adaptation period): November 25th, 1998
- 1st dosing: November 30th, 1998
- Study termination in-life phase males: December 28th, 1998 / females: January 21st, 1999
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
225 mg/kg bw/day (nominal)
Remarks:
Intermediate dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
10 animals for sex for dose (total animals for the study 80). A sufficient number in order to ensure at least 8 pregnant female rats/group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected by the sponsor based on toxicological data already available.
- Rationale for animal assignment (if not random): randomly paired for mating.
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS:
Individual animals were observed at least once daily for any signs of behavioural changes, reaction to treatment or illness. Immediately after administration any signs of illness or reaction to treatment were recorded. In case of changes the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.30 a.m. to 4.30 p.m.. On Saturdays and Sundays animals were checked regularly from 8.00 a.m. to 12.00 a.m. with a final check performed at approximately 4.00 p.m..

BODY WEIGHT:
Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 1 post-partum) and day 4 post-partum. Body weights were recorded individually for each adult animal.

FOOD CONSUMPTION AND COMPOUND INTAKE
The quantity of food consumed by each rat was recorded weekly by weighing the residue: in particular for both sexes weekly until pairing for mating mated , for females weekly during pregnancy (gestation days 7, 14, 20) and for females with litter on lactation day 4. The substance was administrated by oral gavage and not in food.

WATER CONSUMPTION AND COMPOUND INTAKE
Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study. The substance was administrated by oral gavage and not directly in drinking water.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day: dams with offspring were sacrificed on day 4 post-partum, or shortly thereafter. Females showing no evidence of copulation were sacrificed 24-26 days after the last day of the mating period. The male animals were sacrificed after a minimum total dosing period of 28 days if no longer needed for further mating.
- Organs examined: the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the reproductive organs (ovaries, testes, epididymides and accessory sex organs).

MORTALITY:
Checks were made early in each working day and again in the afternoon to look for dead or moribund animals. This would have allowed postmortem examinations to be carried out during the working period of that day. On Saturdays and Sundays a similar procedure was followed except that the final check was carried out at approximately mid-day.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination.
Examinations included: number of corpora lutea, number of implantations, pre-implantation loss %, post-implantation loss %.
Fetal examinations:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups) and the presence of gross abnormalities. Live pups were counted and the sex was determined. Dead pups and pups killed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.
Statistics:
For all numerical values homogeneity of variances was tested using the Bartlett chisquare test. When the variances were homogeneous, the Dunnett test (p ≤ 0.01) was used to compare the experimental groups with the control group. In case of heterogeneity of variances, the Student's t-test was carried out, limit of significance was p ≤ 0.01. For the comparison of classification measurements the FlSHER’s exact test (n < 100) or chi2-test with Yates’ correction for continuity (n ≥ 100) (p ≤ 0.05 and p ≤ 0.01) were employed.
Indices:
For each group the following reproductive indices were determined: gestation index, fertility index female (%), fertility index male (%), pre-implantation loss (%) and post-implantation loss(%). For each litter and group the following indices were determined: birth index, live birth index and viability index.
Clinical signs:
no effects observed
Description (incidence and severity):
No changes of behaviour and external appearance were noted in the female parent animals after treatment with 50, 225 or 1000 mg/kg bw/ day.
Mortality:
no mortality observed
Description (incidence):
None of the low-, intermediate- and high-dosed female parent animals died prematurely.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight change were within the normal range of the control in the male rats treated with 50, 225 or 1000 mg/kg bw/day during the 2-week premating phase and the further 2 test weeks. Also for the females the body weight and body weight gain were within the normal range of the control at all dosages tested and during all phases of the study (premating, mating, pregnancy and lactation period).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No substance-related influence was found on absolute and relative food consumption compared to the control at 50, 225 or 1000 mg/kg bw/day. The slight but statistically significant decrease (at p ≥ 0.01) noted in the relative food consumption in all substance-treated dams (50, 225 or 1000 mg/kg bw/day) during the first gestation week is considered to lie within the normal variability, most likely due to slightly above average food consumption in the control group. Hence the effect was not considered to be substance-related. The absolute and relative food consumption remained unchanged compared to the control during premating, mating, pregnancy weeks 2 and 3 as well as during the lactation period. The substance was administrated by oral gavage and not in food.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Drinking water consumption was not influenced as observed during daily visual appraisal. The substance was administrated by oral gavage and not directly in drinking water.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
None of the female parent animals showed any substance-related pathological findings at necropsy. However, an increased pre-implantation loss (5.0%) was noted at the high dose of 1000 mg/kg (control 2.0%).
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological examination of ovaries of the rats dosed with 1000 mg/kg bw revealed no substance-related changes in any of the female parent animals.
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions observed. Only one female animal (no. 34 at 50 mg/kg bw/day) did not become pregnant.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day the number of pups at birth and during the 4-day lactation period was slightly decreased compared to the control correlating with the reduced number of implants. Hence the mean post-implantation loss was increased to 13.2% (control: 5.9 %) and the mean birth index and live birth index (both: 86.8 %) were decreased compared to the control indices (both: 94.1%). However, the mean viability index (97.6 %) was in the range of the control (93.7 %). Further differences observed in comparison to the control are without any biological relevance.
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of pups alive was not influenced for any dose.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
For all doses the duration of pregnancy of all the other animals was within the normal range of the control, the parturition was not influenced.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): For all doses the duration of pregnancy of all the other animals was within the normal range of the control, the parturition was not influenced.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Mean precoitaltime and duration of pregnancy were within the normal range, the parturition was not influenced. Only one female animal (no. 34 at 50 mg/kg bw/day) did not become pregnant.
Dose descriptor:
NOEL
Effect level:
ca. 225 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: increased pre-implantation loss at 1000 mg/kg bw/ day
Remarks on result:
other: increased pre-implantation loss at 1000 mg/kg bw/ day
Abnormalities:
effects observed, treatment-related
Localisation:
other: increased pre-implantation loss at 1000 mg/kg bw/ day
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The body weight of pups on the 1st and the 4th lactation day was within the range ofthe control.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
At 50 or 225 mg/kg bw/day the number of the pups alive was not influenced. At 1000 mg/kg bw/day the number of pups at birth and during the 4-day lactation period was slightly decreased compared to the control correlating with the reduced number of implants.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the pups was not influenced at any dose.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The body weight of pups on the 1st and the 4th lactation day was within the range ofthe control. Presence of milk was noted in the stomach of almost all pups with only a few exceptions.
External malformations:
no effects observed
Description (incidence and severity):
Stillbirths or malformed pups did not occur at any dose.
Dose descriptor:
NOEL
Effect level:
ca. 225 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced number of pups at 1000 mg/kg bw/day due to the reduced number of implants.
Remarks on result:
other:
Remarks:
reduced number of pups at 1000 mg/kg bw/day due to the reduced number of implants.
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: reduced number of pups at 1000 mg/kg bw/day
Developmental effects observed:
no
Conclusions:
NOEL of similar substance 1 (developmental toxicity) = 225 mg/kg bw /day equivalent to NOAEL Tris(ethyl acetoacetato-O1',O3)aluminium (developmental toxicity) = 239 mg/kg bw/day.
Executive summary:

The substance was tested to evaluate the effect on reproduction and/or development on Sprague-Dawley rats after oral administration by gavage.Ten animals were treated per sex and dose groups resulting in a total of 80 animals. Dose levels were 0, 50, 225 and 1000 mg/kg bw/day. Daily treatment was performed from 2 weeks before mating to the end of the mating period (for the males) and to the 4th day of lactation (for females). There were no effects on mortality, clinical signs, body weights and food consumption. There were some effects in the highest dose groups: the number of pups at birth and during the 4-day lactation period was slightly decreased compared to the control correlating with the reduced number of implants but the mean viability index was in range of control. Based on the reduced number of pups at 1000 mg/kg bw/day NOEL for developmental toxicity of the test item was set as follows: NOEL of similar substance 1 (developmental toxicity) = 225 mg/kg bw /day and the recalculated NOAEL for Tris(ethyl acetoacetato-O1',O3)aluminium is 239 mg/kg bw/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 27th, 1995
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Similar substance 1 of Tris(ethyl acetoacetato-O1’,O3)aluminium
IUPAC Name:
Similar substance 1 of Tris(ethyl acetoacetato-O1’,O3)aluminium
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl: CD®BR
Details on species / strain selection:
The rat is a commonly used rodent species for such studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Females (if applicable) nulliparous and non-pregnant: yes (virgin animals)
- Age at study initiation: 68 to 72 days (age at start of adaptation)
- Weight at study initiation: males: 264 - 287 g; females: 195 - 216 g
- Housing: singly in MAKROLON cages type lll (except during the mating period). Granulated textured wood was used as bedding material in the cages. The cages were cleaned and changed once a week. Periodic analysis of the bedding material is conducted at least once a year. For identification, each rat received a continuous number between 1 and 80: a pattern of points was set on paws and/or tail by tattoo, additionally, the animal cages were labelled with the tattooed serial number, sex, study code number, type of study, route of administration, dose level, date of conception and dates of administration.
- Diet: ALTROMIN 1314 ad libitum. Samples of the food are analysed for contaminants twice a year.
- Water: tap water ad libitum. Samples of the drinking water are taken periodically and periodic analyses are performed.
- Acclimation period: 5 acclimatisation days prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2 °C
- Humidity (%): relative humidity 50 % ± 15 %
- Photoperiod (hrs dark / hrs light): the rooms were alternately lit (about 150 lux at approximately 1.5 m room height) and darkened in a 12 hours dark/12 hours light cycle.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: the test substance was suspended in tap water and was administered orally at a constant volume of 10 ml/kg bw 7 days per week during all duration of treatment (different for males and females). The control animals received 10 ml tap water/kg bw/day in the same manner. The test substance preparations were freshly prepared every day. The daily dose of the test substance was adjusted to the animal's body weight daily.
Details on mating procedure:
- M/F ratio per cage: 1 male and 1 female animal were placed per cage (monogamous mating)
- Length of cohabitation: maximum 2 weeks.
- Proof of pregnancy: each morning the females were examined for the presence of sperm. The day of conception (day 0 of pregnancy) was considered to be the day on which sperm was found.
- After 2 weeks of unsuccessful pairing replacement of first male by another male with proven fertility.This procedure was repeated until at least 8 pregnant dams were available for each group.
- After successful mating each pregnant female was caged (how): singly in MAKROLON cages type lll .
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The amount of test item in aqueous samples obtained during the study was determined applying gas chromatography with flame ionization detection (GC/FID). The samples were analysed after addition of an internal standard and liquid/liquid extraction with ethyl acetate. The extracts were diluted and analysed by GC/FID. 18 samples from the aqueous application solutions were withdrawn at the start of the application phase (test day 1) for the determination of the stability, the homogeneity and the test substance concentration. Further, 3 samples were taken from the application solutions at the end of the application phase (test day 40) for the determination of the test substance concentration (total of 21 samples).

CHROMATOGRAPHY CONDITION:
- Column: HP-1 (100% methyl silicone), 25 m, 0.32 mm i.d., 0.52 /Jm film (Hewlett-Packard)
- Injector: 220 °C
- Detector: FID, 280 °C
- Oven: hold 50 °C (2 min.), heat up to 150 °C (20 °C/min.), hold 150 °C (2 min.)
- Carrier gas: Helium, 2.5 ml/min
- Split: 51 ml/min.
- Septum purge: 2.6 ml/min.
- Retention times: test item: approx. 4.7 min. Internal standard: approx. 3.9 min

PRECISION: coefficient of variation of 1.54 % (peak area ratios) indicated a stable and reliable GC system.

LINEARITY: r2= 0.998 : the method proved to be linear over the entire measuring range.

ACCURACY AND PRECISION: accuracy and precision were within 1-10 % (% of nominal concentration or coefficient of variation of the results of repeated analyses).

LOD = 0.654 mg/ml

CONCLUSION: the actual concentrations of the samples were within the range of 91.8 - 110.0 % of the nominal etest item concentrations indicating correctly prepared, homogenous and stable mixtures.
Duration of treatment / exposure:
Males: 2 weeks prior to mating, during the mating period and approx. 2 weeks post mating at least until the minimum total dosing period of 28 days had been completed (up to and including the day before sacrifice).
Females: throughout the study beginning 2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.
Frequency of treatment:
Daily 7 days per week for all the duration of treatment (different for males and females).
Details on study schedule:
- Start of the study (adaptation period): November 25th, 1998
- 1st dosing: November 30th, 1998
- Study termination in-life phase males: December 28th, 1998 / females: January 21st, 1999
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
low dose
Dose / conc.:
225 mg/kg bw/day (nominal)
Remarks:
intermediate dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
high dose
No. of animals per sex per dose:
10 animals for sex for dose (total animals for the study 80). A sufficient number in order to ensure at least 8 pregnant female rats/group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected by the sponsor based on toxicological data already available.
- Rationale for animal assignment (if not random): randomly paired for mating.
Positive control:
Not present

Examinations

Parental animals: Observations and examinations:
MORTALITY:
Checks were made early in each working day and again in the afternoon to look for dead or moribund animals. This would have allowed postmortem examinations to be carried out during the working period of that day. On Saturdays and Sundays a similar procedure was followed except that the final check was carried out at approximately mid-day.

DETAILED CLINICAL OBSERVATIONS:
Individual animals were observed at least once daily for any signs of behavioural changes, reaction to treatment or illness. Immediately after administration any signs of illness or reaction to treatment were recorded. In case of changes the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.30 a.m. to 4.30 p.m.. On Saturdays and Sundays animals were checked regularly from 8.00 a.m. to 12.00 a.m. with a final check performed at approximately 4.00 p.m..

BODY WEIGHT:
Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 1 post-partum) and day 4 post-partum. Body weights were recorded individually for each adult animal.

FOOD CONSUMPTION AND COMPOUND INTAKE
The quantity of food consumed by each rat was recorded weekly by weighing the residue: in particular for both sexes weekly until pairing for mating mated , for females weekly during pregnancy (gestation days 7, 14, 20) and for females with litter on lactation day 4. The substance was administrated by oral gavage and not in food.

WATER CONSUMPTION AND COMPOUND INTAKE
Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study. The substance was administrated by oral gavage and not directly in drinking water.

OTHER: The duration of gestation of the female rats was recorded and was calculated from day 0 of gestation.
Oestrous cyclicity (parental animals):
The duration of pregnancy was measured and ovaries were weighed to evaluate the effects of test item on female reproductive performance.
Sperm parameters (parental animals):
The preicoital time was measured and the testes and epididymides of all male adult animals were weighed to evaluate the effects of test item on male reproductive performance.
Litter observations:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups) and the presence of gross abnormalities. Live pups were counted and the sex was determined. The pups were weighed on days 1 and 4 post-partum. Any abnormal behaviour of the pups was recorded.

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: the male animals were sacrificed after a minimum total dosing period of 28 days if no longer needed for further mating.
- Maternal animals: dams with offspring were sacrificed on day 4 post-partum, or shortly thereafter. Females showing no evidence of copulation were sacrificed 24-26 days after the last day of the mating period.

GROSS NECROPSY
The adult animals were examined macroscopically for any abnormalities or pathological changes and special attention was paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The testes and epididymides of all male adult animals were weighed. The ovaries, testes, epididymides, accessory sex organs and all organs showing macroscopic lesions of all adult animals were preserved. The testes and epididymides were preserved in Bouin’s fixative; the remaining tissues were preserved in 7 % buffered formalin. Detailed histological examination was performed on the following organs of all animals of groups 1 (control group) and 4 (high dose level group) after preparation of paraffin sections and haematoxylin-eosin staining: ovaries, testes, epididymides.

Postmortem examinations (offspring):
Dead pups and pups killed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.

Statistics:
For all numerical values homogeneity of variances was tested using the Bartlett chisquare test. When the variances were homogeneous, the Dunnett test (p ≤ 0.01) was used to compare the experimental groups with the control group. In case of heterogeneity of variances, the Student's t-test was carried out, limit of significance was p ≤ 0.01. For the comparison of classification measurements the FlSHER’s exact test (n < 100) or chi2-test with Yates’ correction for continuity (n ≥ 100) (p ≤ 0.05 and p ≤ 0.01) were employed.
Reproductive indices:
For each group the following reproductive indices were determined: gestation index, fertility index female, fertility index male, pre-implantation loss and post-implantation loss.
Offspring viability indices:
For each litter and group the following indices were determined: birth index, live birth index and viability index.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No changes of behaviour and external appearance were noted in the male and female parent animals after treatment with 50, 225 or 1000 mg/kg bw/day.

Mortality:
no mortality observed
Description (incidence):
None of the low-, intermediate- and high-dosed male and female parent animals died prematurely.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight change were within the normal range of the control in the male rats treated with 50, 225 or 1000 mg/kg bw/day during the 2-week premating phase and the further 2 test weeks. Also for the females the body weight and body weight gain were within the normal range of the control at all dosages tested and during all phases of the study (premating, mating, pregnancy and lactation period).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No substance-related influence was found on absolute and relative food consumption compared to the control at 50, 225 or 1000 mg/kg bw/day. The slight but statistically significant decrease (at p ≥ 0.01) noted in the relative food consumption in all substance-treated dams (50, 225 or 1000 mg/kg bw/day) during the first gestation week is considered to lie within the normal variability, most likely due to slightly above average food consumption in the control group. Hence the effect was not considered to be substance-related. The absolute and relative food consumption remained unchanged compared to the control during premating, mating, pregnancy weeks 2 and 3 as well as during the lactation period. The substance was administrated by oral gavage and not in food.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Drinking water consumption was not influenced as observed during daily visual appraisal. The substance was administrated by oral gavage and not directly in drinking water.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological examination of testicle, epididymis and ovaries of the rats dosed with 1000 mg/kg bw revealed no substance-related changes in any of the male and female parent animals.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The duration of pregnancy of all the other animals was within the normal range of the control, the parturition was not influenced.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Pre-coital time was similar in both control animals and rats treated with 50, 225 or 1000 mg/kg bw/day.
Reproductive performance:
effects observed, non-treatment-related

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed
Remarks on result:
other: no adverse effects observed
Dose descriptor:
NOEL
Effect level:
ca. 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: increased pre-implantation loss
Remarks on result:
other: increased pre-implantation loss

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
At 50 or 225 mg/kg bw/day the number of the pups alive was not influenced. At 1000 mg/kg bw/day the number of pups at birth and during the 4-day lactation period was slightly decreased compared to the control correlating with the reduced number of implants.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight of pups on the 1st and the 4th lactation day was within the range of the control. Presence of milk was noted in the stomach of almost all pups with only a few exception
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The body weight of pups on the 1st and the 4th lactation day was within the range of the control. Presence of milk was noted in the stomach of almost all pups with only a few exception

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
ca. 225 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced number of pups at 1000 mg/kg bw/day due to the reduced number of implants.
Remarks on result:
other: reduced number of pups at 1000 mg/kg bw/day due to the reduced number of implants.

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOEL of similar substance 1 (reproductive toxicity) > 1000 mg/kg bw/day (males) and NOEL of similar substance 1 (reproductive toxicity) = 225 mg/kg bw/day (females) equivalent to NOAEL of Tris(ethyl acetoacetato-O1',O3)aluminium (reproductive toxicity) > 1062 mg/kg bw/day (males) and NOAEL of Tris(ethyl acetoacetato-O1',O3)aluminium (reproductive toxicity) = 239 mg/kg bw/day (females).
Executive summary:

The substance was tested to evaluate the effect on reproduction and/or development on Sprague-Dawley rats after oral administration by gavage. Ten animals were treated per sex and dose groups resulting in a total of 80 animals. Dose levels were 0, 50, 225 and 1000 mg/kg bw/day. Daily treatment was performed from 2 weeks before mating to the end of the mating period (for the males) and to the 4th day of lactation (for females).There were no effects on mortality, clinical signs, body weights and food consumption. There were some effects in the highest dose groups: the number of pups at birth and during the 4-day lactation period was slightly decreased compared to the control correlating with the reduced number of implants but the mean viability index was in range of control. Based on the increased pre-implantation loss at 1000 mg/kg bw/day NOEL for reproductive toxicity of similar substance 1 was set as follows: NOEL > 1000 mg/kg bw/day (males) and NOEL= 225 mg/kg bw/day (females). The recalculated NOAEL reproductive toxicity for Tris(ethyl acetoacetato-O1',O3)aluminium is > 1062 mg/kg bw/day (males) and equal to 239 mg/kg bw/day (females).