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Toxicological information

Developmental toxicity / teratogenicity

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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:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd
- Age at study initiation: Approximately 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°C
- Humidity: 55 ± 15%
- Photoperiod: 12 h light / 12 h dark
Route of administration:
oral: gavage
Vehicle:
other: MOL WO M 46 Medicinal white oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle: 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.: 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°C in the dark for 13 days, by IR spectroscopy using a Perkin Elmer Spectrum One Fournier-transform infrared spectrophotometer.
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
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Basis: Dose (test item in vehicle)
Dose / conc.:
375 mg/kg bw/day (nominal)
Remarks:
Basis: Dose (test item in vehicle)
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
Basis: Dose (test item in vehicle)
Dose / conc.:
1 500 mg/kg bw/day (nominal)
Remarks:
Basis: Dose (test item in vehicle)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Basis: Concentration of test item (active ingredient)
Dose / conc.:
56.3 mg/kg bw/day (actual dose received)
Remarks:
Basis: Concentration of test item (active ingredient)
Dose / conc.:
113 mg/kg bw/day (actual dose received)
Remarks:
Basis: Concentration of test item (active ingredient)
Dose / conc.:
225 mg/kg bw/day (actual dose received)
Remarks:
Basis: Concentration of test item (active ingredient)
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: 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
Positive control:
Not included
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS
- Time schedule: Multiple occasions during each day for morbidity and mortality
- Cage side observations recorded: Yes

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

BODY WEIGHT
- 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
- Time schedule for examinations: 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
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

FOOD EFFICIENCY
- Time schedule for examinations: 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.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY
- 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
- 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
- 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
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Pituitary
Bone & bone marrow (sternum)
Prostate
Brain (including cerebrum, cerebellum and pons)
Oesophagus
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
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.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths and no toxicologically significant clinical observations detected.
BODY WEIGHT AND WEIGHT GAIN
There were no toxicologically significant effects detected on body weight development.
FOOD CONSUMPTION
No adverse effect on food consumption was deteted in treated animals
FOOD EFFICIENCY
No adverse effect on food efficiency was detected in treated animals
WATER CONSUMPTION
No adverse effect on water consumption was detected
OPHTHALMOSCOPIC EXAMINATION
Not examined
HAEMATOLOGY
No toxicologically significant effects were detected
CLINICAL CHEMISTRY
No toxicologically significant effects were detected
URINALYSIS
Not examined
NEUROBEHAVIOUR
There were no toxicologically significant changes in the behavioural paremeters measured, in functional performance or sensory reactivity
ORGAN WEIGHTS
No toxicologically signifcant treatment related trends were detected in the organ weights measured.
GROSS PATHOLOGY
There were no macroscopic abnormalities detected that were considered to be related to treatment
HISTOPATHOLOGY
No treatment related microscopic findings were detected
Key result
Dose descriptor:
NOAEL
Effect level:
> 225 other: mg/kg bw/day Active Ingredient
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects seen on clinical signs; mortality; body weight; food consumption; water consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathlogy at doses up to 1500 mg/kg bw/day (225 mg/kg bw/day Active Ingredient)
Critical effects observed:
not specified
Conclusions:
The subchronic repeated dose oral toxicity of grease containing 15% active ingredient (aluminum, benzoate C16-18-fatty acids complexes) in MOL WO M 46 medicinal white oil to rats gave a NOAEL of 1500 mg/kg bw/day (225 mg/kg bw/day Active Ingredient).
Executive summary:

The subacute repeated dose oral toxicity of grease containing 15% active ingredient (aluminum, benzoate C16-18-fatty acids complexes) in MOL WO M 46 medicinal white oil to rats gave a NOAEL of 1500 mg/kg bw/day (225 mg/kg bw/day Active Ingredient)

The repeated dose oral toxicity of aluminum, benzoate C16 -18 -fatty acids complexes to rats is taken from data presented within an oral (gavage) combined repeat dose toxiciy study with reproduction/developmental toxicity screening test in the rat performed in compliance with, amongst others, the requirements of the OECD Guidelines for Testing of Chemicals No.422 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Screening Test" (adopted 22 March 1996). Since the results are taken from a regulatory and GLP compliant study, the data are considered reliable and relevant for use in assessing this endpoint.

Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd
- Age at study initiation: Approximately 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°C
- Humidity: 55 ± 15%
- Photoperiod: 12 h light / 12 h dark
Route of administration:
oral: gavage
Vehicle:
other: MOL WO M 46 Medicinal white oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle: 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.: 9037038
- Purity: 100%
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)
- Post-mating: After successful mating each pregnant female was caged individually and allowed to give birth and maintain their offspring until Day 5 post partum.
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°C in the dark for 13 days, by IR spectroscopy using a Perkin Elmer Spectrum One Fournier-transform infrared spectrophotometer.
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
Details on study schedule:
- Age at mating of the mated animals in the study: 14 weeks
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Basis: Dose (test item in vehicle)
Dose / conc.:
375 mg/kg bw/day (nominal)
Remarks:
Basis: Dose (test item in vehicle)
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
Basis: Dose (test item in vehicle)
Dose / conc.:
1 500 mg/kg bw/day (nominal)
Remarks:
Basis: Dose (test item in vehicle)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Basis: Concentration of test item (active ingredient)
Dose / conc.:
56.3 mg/kg bw/day (actual dose received)
Remarks:
Basis: Concentration of test item (active ingredient)
Dose / conc.:
113 mg/kg bw/day (actual dose received)
Remarks:
Basis: Concentration of test item (active ingredient)
Dose / conc.:
225 mg/kg bw/day (actual dose received)
Remarks:
Basis: Concentration of test item (active ingredient)
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: 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
Positive control:
Not included
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS
- Time schedule: Multiple occasions during each day for morbidity and mortality
- Cage side observations recorded: Yes

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

BODY WEIGHT
- 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
- Time schedule for examinations: 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
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

FOOD EFFICIENCY
- Time schedule for examinations: 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.

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
- 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
Oestrous cyclicity (parental animals):
Not determined
Sperm parameters (parental animals):
- Parameters examined in P male parental generations: Organ weights and histopathological examination of testes, epididymides and seminal vesicles
Litter observations:
Litters were examined as soon as possible after delivery (LD 0) and on LD 4.

PARAMETERS EXAMINED
- The following parameters were examined in F1 offspring: Litter size, number of stillborn and liveborn pups, survival, number of males and females, individual body weights, abnormal behaviour, and gross abnormalities of the pups, surface righting reflex on Day 1 post partum

GROSS EXAMINATION OF DEAD PUPS: Yes, for external abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on Day 43
- Maternal animals: All surviving animals on Day 5 post partum

GROSS NECROPSY: Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS: The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring sacrificed on LD 4.
- These animals were subjected to postmortem external examinations.

GROSS NECROPSY
- Gross necropsy consisted of external examinations.
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.
Reproductive indices:
MATING PERFORMANCE AND FERTILITY
The following parameters were calculated from the individual data during the mating period of the parental generation:
i) Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = x 100
Pregnancy Index (%) = x 100

GESTATION AND PARTURITION DATA
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
i) Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii) Parturition Index: The following was calculated for each group: Parturition Index (%) = x 100
Offspring viability 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
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no unscheduled deaths and no significant clinical observations detected.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no significant effects on bodyweight development and no adverse effect on food consumption or food utilisation efficiency
REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no indications of any changes to the morphology or function of the male gonads as indicated by no effects on oragn weights or pathology of the testes, epididymides and seminal vesicles.
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no efects recorded on mating performance, fertility indices or gestation length.
ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no significant effects detected in the organ weights measured
GROSS PATHOLOGY (PARENTAL ANIMALS)
No significant macroscopic abnormalities were detected
HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment related microscopic findings were detected
OTHER FINDINGS (PARENTAL ANIMALS)
There were no significant changes detected in the behavioural parameters measured or the functional performance of adult animals and there were no treatment related changes in sensory reactivity
Key result
Dose descriptor:
NOAEL
Effect level:
> 225 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Reproductive parameters (male/female fertility and fecundity indices, and copulatory intervals) were unaffected by treatment. No statistically significant changes in delivery data and pup development/survival.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
No significant differences were detected for litter viability for treated animals when compared to controls.
CLINICAL SIGNS (OFFSPRING)
No significant effects were observed
BODY WEIGHT (OFFSPRING)
No significant differeces in litter size were detected between controls and treated animals.
OTHER FINDINGS (OFFSPRING)
There were no significant effects on surface righting reflex for treated animals when compared with the controls.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 225 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No reproductive effects or definitive test article related changes in the development or survival of the offspring.
Key result
Reproductive effects observed:
no
Conclusions:
Oral administration of aluminum, benzoate C16-18 fatty acid complexes to parental 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) 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:

Oral administration of aluminum, benzoate C16-18 fatty acid complexes to parental 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) did not result in any adverse reproductive effects or any definitive test article-related changes in the development or survival of the offspring.

Results from the clinical pathology evaluations and neurobehavioral testing on parent animals did not reveal any definitive effects that could be attributed to treatment at the dose levels tested. In addition there were no changes in blood chemisry and haematlogy assessments of parent animals that were indicative of a reaction to treatment. Consequently, in the absence of any pathological and oragn weight changes amongst the parent animals there was no eveidence of any systemic effects at any of the dose levels examined.

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: Approximately 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 °C
- Humidity: 55 ± 15%
- Photoperiod: 12 h light / 12 h 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: 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.: 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°C 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)
- Post-mating: 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: 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
- Time schedule: Multiple occasions during each day for morbidity and mortality
- Cage side observations recorded: Yes

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

BODY WEIGHT
- 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
- Time schedule for examinations: 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
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

FOOD EFFICIENCY:
- Time schedule for examinations: 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.

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:
- 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
- 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, 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
Remarks:
Generation F1
Effect level:
> 225 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No reproductive effects or definitive test article related changes in the development of survival of the offspring.

Overall developmental toxicity

Key result
Developmental effects observed:
no

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 (Harlan 2013). 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.