Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as an unpublished report.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Deviations:
not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material: Aluminum, benzoate C16-18 fatty acid complexes
- Substance type: technical product
- Physical state: pale yellow solid
- Batch number: 11074091 + benzoic acid + white oil
- Date recieved: 07 July 2012
- Expiry date: 01 July 2013
- Storage conditions: Room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- source: Harlan Laboratories UK Ltd
- age at study initiation: approx 12 weeks
- weight at study initiation: males - 322 to 394g; females (nulliparous and nonpregnant) - 195 to 237g
- housing: initially in groups of 4 in solid floor propylene cages with softwood bedding. During pairing animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbant paper, one male:one female basis. Following successful mating, males returned to original cages. Mated females housed individually during gestation/lactation in the solid floor cages as for mating. Enrichment: wooden chew blocks and cardboard tunnels.
-diet: Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories UK Ltd
- water: mains drinking water ad libitum
- acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- temperature: 21 +/- 2 deg C
- humidity: 55+/- 15%
- photoperiod: 12h light/12h dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: MOL WOM46 Medicinal white oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): test material synthesised in the presence of MOL WO M 46 Medicinal white oil. Same white oil used for dilution of test material and as the control vehicle
- Test substance concentration in vehicle: 15%
- Treatment volume: 5 ml/kg bw/day
- Lot/batch no. (if required): 9037038
- Purity: 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the test material in the vehicle dilutions were determined by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). The test item formulations were extracted with hexane, evaporated to dryness and re-dissolved in 2% nitric acid. Homogeneity determinations were performed on samples taken from the top, middle and bottom of the container. Stability determinations were performed before and after storage for 13 days at approx +4 degC in the dark for 13 days, by IR spectroscopy using a Perkin Elmer Spectrum One Fournier-transform infrared spectrophotometer.
Details on mating procedure:
- M/F ratio per cage: 1 male to 1 female within each dose group
- Length of cohabitation: Maximum of 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as day 0 of pregnancy (post coitum)
- After successful mating each pregnant female was caged individually and allowed to give birth and maintain their offspring until Day 5 post partum.
Duration of treatment / exposure:
Males dosed for 42 days and killed on day 43, beginning 14 days prior to mating.
Dosing of females began 14 days before mating, and continued through mating, up to and including day 4 post-partum. They were killed on day 5 post-partum.
Frequency of treatment:
Daily, once per day
Duration of test:
Up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation).
No. of animals per sex per dose:
12 males and 12 females per dose level
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected on the basis of a 14-day dose range finding study where three groups of 3 male and 3 female wistar rats were treated at 375, 750 and 1500 mg/kg bw/day (dosed as supplied, containing 15% active ingredient). A group of 3 males and 3 females received the vehicle (medicinal white oil). No signs of toxicity were observed, and no adverse effects on bodyweight, food consumption, or water consumption. No macroscopic changes were seen at necropsy.
- Rationale for animal assignment (if not random): The animals were allocated to dose groups using a randomised procedure based on stratified bodyweights. Group mean bodyweights were then dermined to ensure similarity between the groups.
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: multiple occasions during each day for morbidity and mortality
- Cage side observations recorded

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before dosing, 30 mins, 1 and 5h after dosing during weekdays; before dosing and 1h after dosing at weekends

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing, then weekly for males until termination, and weekly for females until mating was evident. Then for females bodyweight was recorded on days 0, 7, 14 and 20 post coitum, and on days 1 and 4 post partum.

FOOD CONSUMPTION:
- Food consumption was recorded for each cage of adults and was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

WATER CONSUMPTION:
Water intake was observed daily by visual inspection of water bottles for any overt changes.

FOOD EFFICIENCY:
- Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 42 for males, day 4 post partum for females
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 5 males and 5 females per group
- Parameters checked:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 42 for males, day 4 post partum for females
- Animals fasted: No
- How many animals: 5 males and 5 females per group
- Parameters checked
Urea Calcium (Ca++)
Glucose Inorganic phosphorus (P)
Total protein (Tot.Prot.) Aspartate aminotransferase (ASAT)
Albumin Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation) Alkaline phosphatase (AP)
Sodium (Na+) Creatinine (Creat)
Potassium (K+) Total cholesterol (Chol)
Chloride (Cl-) Total bilirubin (Bili)
Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:prior to start of treatment and weekly intervals thereafter. Functional performance tests performed on 5 selected males and females from each dose level prior to termination, together with an assessment of sensory reactivity to various stimuli.
-Behavioural assessments: Detailed individual clinical observations were performed for each animal using a purpose built arena. This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioural Assessments and Sensory Reactivity Tests.
- Functional/performance tests: motor activity, forelimb/hindlimb grip strength
- Sensory reactivity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).
- Dose groups that were examined: each group
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data not analysed by the Provantis data capture system were assessed separately using the SPSS statistical package. Initially, the homogeneity of the data was assessed using Levene’s test. Where Levene’s test was shown to be non-significant (p≥0.05), parametric analysis of the data was applied, incorporating analysis of variance (ANOVA). If this data was shown to be significant, this analysis was followed by pair-wise comparisons using Dunnett’s test. Where Levene’s test was significant, non-parametric analysis of the data was analysed incorporating the Kruskal-Wallis test which if significant, was followed by the Mann-Whitney U test. Dose response relationship was also be investigated by linear regression. Where the data was unsuitable for these analyses, then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Due to the preponderance of non-normally distributed data, reproductive parameters (implantation losses, offspring sex ratio and offspring surface righting) were analysed using non-parametric analyses.
Indices:
i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
Pre–implantation loss = x100
Post–implantation loss = x 100
ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = x 100
Viability Index (%) = x 100
iii) Sex Ratio (% males)
Sex ratio was calculated for each litter on Days 1 and 4 post partum, using the following formula: x 100
Historical control data:
No data reported

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Remarks:
Generation P male/female
Effect level:
> 225 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 225 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Oral administration of aluminum, benzoate C16-18 fatty acid complexes to male and female rats at dose levels of 375, 750 and 1500 mg/kg bw/day (equivalent to 56.3, 113 and 225 mg/kg bw/day Active Ingredient) did not result in any adverse reproductive effects or any definitive test article-related changes in the development or survival of the offspring.
Executive summary:

The developmental toxicity of aluminum, benzoate C16 -18 fatty acid complexes was assessed in a combined repeated dose and reproductive toxicity screening test following OECD guideline 422 (MPI 2011). The parental generation was dosed by daily oral gavage each day with aluminum, benzoate C16 -18 fatty acid complexes at dose levels of 375, 750 and 1500 mg/kg bw/day (equivalent to 56.3, 113 and 225 mg/kg bw/day Active Ingredient). The offspring of the treated rats were then assessed for survival (gestation and postnatal survival indices, percent pre- and post-implantation loss), pup body weight and sex ratio and external abnormalities.

There were no effects seen on any of the developmental toxicity parameters measured and there were no indications of any systemic effects at any dose level.