Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Data are taken from a read-across oral reprotoxicity screening study in rats with aluminum, benzoate C16-18 fatty acid complexes, and conducted according to OECD 422, in which no adverse effect was seen in any of the reproductive parameters examined at any dose.

Link to relevant study records

Referenceopen allclose all

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.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
exposure considerations
Justification for data waiving:
the study does not need to be conducted because (i) the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), (ii) it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and (iii) there is no or no significant human exposure
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
REACH Annex IX Column 1 states that a further reproductive toxicity study is required if the 28-day (or 90-day) study indicates an adverse effect on reproductive organs or tissues. Since no toxicity of this type was seen in the OECD422 study with aluminum, benzoate C16-18 fatty acid complexes, then the Annex IX requirement for a further reproduction study for the registered substance is waived.

An OECD 422 study was conducted in rats by daily oral gavage administration of aluminium, benzoate, C16-18 fatty acids complexes manufactured in situ in a medicinal grade white oil base and the results have been read across to the substance. There were no effects on any of the reproductive or developmental parameters measured. As the maximum dose that could be administered was limited by the physico-chemical characteristics of the dosing preparation, the limit dose of actual aluminium, benzoate C16-18 fatty acid complexes could not be attained and the maximum dose was below the limit dose of 1500 mg/kg/day (actual maximum dose achieved was 225 mg/kg bw/day, which was considered to be the NOAEL).

The registered substance is manufactured and used solely in situ in an inert carrier. The registered substance and aluminium, benzoate, C16-18 fatty acids complexes are used specifically as thickeners in industrial greases and lubricants and as such typically occur in base oil. The interactions between the thickener and base oil do not strictly fall under the definitions of a reaction product nor do they act as a simple mixture of components due to the matrix effects which occur in the manufacturing/ blending process. Matrix effects should be taken into account when assessing whether adverse effects would be seen in grease products because entrainment of grease thickeners during the manufacturing process as part of the grease matrix severely limits exposure and will have a significant impact on the outcome of any risk assessments. The fact that matrix effects do occur is a point recognised in the OECD Lubricant Emission Scenario document (2004) and it is therefore reasonable to assess the influence the process of manufacturing the thickener in an inert base oil has on factors such as accessibility, which are critical to assessing the potential risks posed by the grease thickeners when in the grease.

Leaching tests have been undertaken to assess whether the metallic components of the metal-soap and metal-complex-soap grease thickeners remain in base grease(s) and hence determine the degree of accessibility of the thickeners (see section 4.6 and Appendix 2 of the CSR). The results of the study show no leaching of the thickener from the base oil and a lack of bioaccessibility of the substance in aqueous media. The same lack of bioaccessibility is expected in GI fluids as well and, as a further demonstration of this lack of accessibility, it is proposed to conduct a leaching study in fed state simulated intestinal fluid (FeSSIF) using the registered substance and the structural analogue aluminium, benzoate, C16-18 fatty acids complexes.

This substance has been registered by a Member of the European REACH Grease Thickeners Consortium (ERGTC). A number of decisions have been made in the dossier with regard to the approach taken for registering the substance including the testing strategy and the justification for waiving certain endpoints. Several of the decisions reflect the technical difficulties of testing the substance and the relevance of data with regard to the potential for exposure, given that the substance typically occurs in situ in base oil. A face to face meeting between the ERGTC and ECHA was held in Helsinki on 8th September 2016 which discussed many of these topics and a copy of the minutes from the meeting are attached to the dossier (See section 13 of IUCLID). Therefore, if there are any queries or concerns which arise when the dossier is reviewed, it is requested that the reviewer discuss these with the ERGTC (ERGTC@wca-consulting.com) as there may be background information and previously discussions between the ERGTC and ECHA available which are relevant.
Reproductive effects observed:
not specified
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with the Regulation (EC) No 1907/2006, Annex XI, section 1.5, read-across to aluminum, benzoate C16-18-fatty acids complexes has been used to fulfil REACH information requirements where appropriate and is justified by the chemical structures and common physiological active moieties of the substances. The chemical structures of the target and read-across substances are very closely aligned. The aluminium cation, a long chain fatty acid, and the –Al=O (-AlOH in aqueous solution) moieties are identical in both substances. The key difference is that read-across substance contains a benzoate moiety linked to the aluminium cation, which is absent from the target substance. Benzoic acid and benzoates have been well characterized (eco)toxicologically, but in this case generating experimental data on the aluminium salt containing benzoate would be expected to demonstrate a ‘worst case’ hazard profile when compared to the target substance. Since no intrinsic toxicity could be demonstrated from any of the Annex VII or VIII endpoints with the benzoate-containing aluminium salt, then these results can be read across to the target substance without restriction.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical: Aluminum, benzoate, C16-18 fatty acids complexes (EC: 303-385-6, CAS: 94166-87-7)

See robust study summaries for further details on the identity of the tested substances and IUCLID dataset for further information on the substance identity and the data to support the read across justification.

3. ANALOGUE APPROACH JUSTIFICATION
Aluminum, benzoate C16-18-fatty acids complexes is considered suitable for read-across as it contains a fatty acid moiety coordinated to an aluminium atom. The chemical structures of the target and read-across substances are very closely aligned; both substances consist of aluminium salts of fatty acids. The aluminium cation, a long chain fatty acid, and the –Al=O (-AlOH in aqueous solution) moieties are identical in both substances.

The fatty acids present in both substances are the same, consisting of a mixture of C16 and C18 chain lengths at approximately a 1:2 ratio. The C16 and C18 fatty acid moieties are derived from natural fatty materials, or substances which are chemically indistinguishable from natural fatty acids. The fatty acid moieties are considered not to be hazardous to humans as they are natural constituents of the human body and essential components of a balanced human nutrition. REACH Annex V, Entry 9, groups fatty acids and their potassium, sodium, calcium and magnesium salts, including C6 to C24, predominantly even-numbered, unbranched, saturated or unsaturated aliphatic monocarboxylic acids. Provided that they are obtained from natural sources and are not chemically modified, the substances included in REACH Annex V, Entry 9 are exempt from registration, unless they are classified as dangerous (except for flammability, skin irritation or eye irritation) or they meet the criteria for PBT/vPvB substances. The fatty acid components of the two substances are therefore expected to be exempt under REACH.

Fatty acids are an endogenous part of every living cell and are an essential dietary requirement. They are absorbed, digested and transported in animals and humans. When taken up by tissues they can either be stored as triglycerides or can be oxidised via the ß-oxidation and tricarboxylic acid pathways. The ß-oxidation uses a mitochondrial enzyme complex for a series of oxidation and hydration reactions, resulting in a cleavage of acetate groups as acetyl CoA. Acetyl CoA is used mainly to provide energy but also to provide precursors for numerous biochemical reactions. Alternative minor oxidation pathways can be found in the liver and kidney (ω-oxidation and ω-1 oxidation) and in peroxisomes for ß-methyl branched fatty acids (α-oxidation). The metabolic products can then be incorporated for example into membrane phospholipids.

Comparison of the data for the two substances indicates that they are expected to have similar properties. Neither the target or read-across substance meets the criteria for classification for physico-chemical, environmental or human health endpoints, based on the available data.

On the basis of the physico-chemical results, the substances are not flammable and have similar densities. The low vapour pressure results indicates that hazards associated with the atmospheric compartment or inhalation routes of toxicity are not expected to be relevant. The substances show similar water solubility, without surface active properties, indicating that they are likely to have similar behaviour in the aquatic environment.

Although the read-across substance met the criteria for ready biodegradability and the target substance (tested as a 50% concentration in pharmaceutical white oil) did not, neither substance was inhibitory to micro-organisms at the concentration tested. The difference in biodegradation results is expected to derive from the presence of the base oil in the target substance sample, which is designed to minimise leaching of the grease thickener, and therefore less of the grease thickener would have been available for degradation by the micro-organisms.

There are no results available for the ecotoxicity of the target substance and therefore comparison of the effect concentrations against the read-across substance is not possible. However, leaching studies on grease thickeners in base oils have been used to assess the potential bioavailability of the grease components. The bioavailability potential of the water accommodated fractions (WAFs) of metal (lithium and calcium) soap complex based grease thickeners was assessed using a solid-phase micro-extraction (SPME) method combined with gas chromatography (GC). This approach was complemented with metal ion analysis to determine whether the metal leaches out of the base grease during WAF preparation and the ecotoxicity of WAFs was also monitored using an in vitro Microtox assay. The SPME-GC data confirmed that there was negligible leaching of the thickeners from base oils in the samples tested, with measurements for calcium and lithium below the limit of detection (<0.1 mg/L) and the screening ecotoxicity data also showed a lack of toxicity of the greases.

The results of the bioavailability potential of the WAFs, the metal ion analysis and the screening ecotoxicity of lithium and calcium based complexes have been read across to aluminium based thickeners. All of these metal salts of fatty acids are expected to behave in a very similar manner when entrained within a grease matrix, with high temperature stability indicating that the thickener structure is robust and resistant to diffusion out of the oil. Dissolution of grease thickeners from grease into water is very unlikely as the thickeners are poorly water soluble and the thickeners are embedded in the hydrophobic grease matrix and thus unlikely to leach out. Therefore, although there are no data on the ecotoxicity of the target substance, no effects are expected based on the lack of bioavailability of the thickener.

These data on the potential for leaching of other metal salt complex based grease thickeners have been read across to both the target and read across substances. On the basis of these results, it is expected that neither the target nor the read across substance would leach from the base oil in which they are typically marketed and therefore neither substance would be bioavailable. Thus, reading across data from the source substance tested in its isolated form is considered robust as it provides a worst-case conclusion for the target substance which is only manufactured in an inert carrier, typically base oil. In order to provide further evidence for the lack of bioavailability, it is proposed to undertake leaching studies on the target and read-across substances themselves. Dependent on the results, the two studies would then be used to show the similarity in the bioavailability of the two substances and provide further weight of evidence for the read-across approach.

The available mammalian toxicity data show that neither the target nor read-across substance would be classified as irritating to skin or eyes and would not be classified for acute oral toxicity, with LD50 values of >2000 mg/kg. Although no other data are available for comparison of the potential mammalian toxicity of the two substances, the target and read-across substances are expected to behave in a very similar manner. As grease thickeners are entrained within grease matrices which are robust and resistant to diffusion out of the oil, neither substance is expected to be bioavailable. In order to provide further evidence for the lack of bioavailability, it is proposed to undertake leaching studies in fed state simulated intestinal fluid (FeSSIF) on the target and read-across substances. Dependent on the results, the two studies would then be used to show the similarity in the bioavailability of the two substances and provide further weight of evidence for the read-across approach.

For the reproductive toxicity of the substances, read across from the source to the target substance is considered justified. Both substances would not leach when in situ in base oil during use as grease thickeners and are not expected to be bioavailable. The substances would dissociate into inorganic aluminium species and fatty acids (plus benzoic acid for the source substance), the organic components of which are readily metabolised. As the fatty acid components are essential nutrients to many organisms and are not expected to be hazardous (and the benzoate component of the source substance is not expected to be hazardous), the toxicity is expected to be driven by the aluminium component, so would be the same in both the source and target substances. As such, read across from the source substance is considered to provide a worst-case scenario for the target substance.

4. DATA

T = target substance (tests were undertaken on a sample prepared as a 50% w.w. concentration in medicinal white oil unless otherwise indicated)
RA = read-across substance

- State: Liquid (T), Solid (RA)
- Melting point: 21°C (T), 224°C (RA)
- Relative density: 0.933 (T), 1.08 (RA)
- Vapour pressure: 0.00015 Pa (T), 0.000044 Pa (RA)
- Surface tension: 72.5 mN/m (T), 72.6 mN/m (RA)
- Water solubility: ≤0.00015 g/L (T), ≤0.00026 g/L (RA)
- Flash-point: 159°C (T), No data available for RA
- Flammability: No data available for T, Not flammable (RA)
- Self-ignition temperature: 374°C (T), 383°C (RA)
- Viscosity: 174.3 mm2/s at 100°C (T), No data available for RA
- Biodegradation: Not readily biodegradable (31%) (T), Readily biodegradable (79%) (RA)
- Acute aquatic invertebrates: No data available for T, EL50 (48 h): > 100 mg/L (RA)
- Algae: No data available for T, EL50 (72 h): > 100 mg/L and NOELR (72 h): 100 mg/L (RA)
- Aquatic microorganisms: NOEC (28 d): 6.7 mg/L (T), NOEC (28 d): 15.4 mg/L (RA)
- Acute fish: No data available for T, LL50 (96 h): > 100 mg/L (RA)
- Skin irritation: Not irritating (T), Not irritating (RA)
- Eye irritation: Not classified (T), Not classified (RA)
- Skin sensitisation: No data available for T, Not sensitising (RA)
- In vitro gene mutation in bacteria: No data available for T, Negative (RA)
- Acute toxicity, oral route: LD50: > 2000 mg/kg (T, test undertaken on solid (isolated) form of the substance), LD50 >2000 mg/kg (RA)
- Acute toxicity, dermal route: No data available for T, LD50 >2000 mg/kg (RA)
- In vitro cytogenicity: No data available for T, Negative (RA)
Reason / purpose:
read-across source
Strain:
Wistar
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 weihts 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:

Aluminium, benzoate, C16 -18 fatty acids complexes is considered suitable for read across as it contains a fatty acid moiety coordinated to an aluminium atom. Although it contains a coordinated benzoate ion, no toxicological effects were observed and therefore it is concluded that the benzoate ion does not contribute any additional toxicity to the substance. Aluminium, benzoate, C16 -18 fatty acids complexes showed no toxicological effects in OECD 422 study (Harlan 2013). Therefore, aluminium benzoate C16 -18 fatty acids complexes is considered to have a NOAEL of 1500 mg/kg bw/day (225 mg/kg bw/day active ingredient) and this has been read across to the target substance.

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
225 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Guideline study conducted to GLP
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Aluminium, benzoate, C16 -18 fatty acids complexes is considered suitable for read across as it contains a fatty acid moiety coordinated to an aluminium atom. Although it contains a coordinated benzoate ion, no toxicological effects were observed and therefore it is concluded that the benzoate ion does not contribute any additional toxicity to the substance. Aluminium, benzoate, C16 -18 fatty acids complexes showed no toxicological effects in OECD 422 study (Harlan 2013). Therefore, aluminium benzoate C16 -18 fatty acids complexes is considered to have a NOAEL of 1500 mg/kg bw/day (225 mg/kg bw/day active ingredient) and this has been read across to the target substance.

A key subacute toxicity and reproductive toxicity screen, using the OECD 422 study design, was conducted in rats on aluminum, benzoate C16-18 fatty acid complexes via oral gavage administration. The test material was administered at dose levels of 0, 375, 750 and 1500 mg/kg bw/day nominal, equating to 56.3, 113 and 225 mg/kg bw/day Active Ingredient. There were no treatment-related effects at any dose level on any of the reproductive parameters evaluated in this study, or in any of the developmental parameters evaluated. Based on these data, the NOAEL for reproductive and developmental toxicity was 1500 mg/kg bw/day, equivalent to 225 mg/kg bw/day Active Ingredient. Data taken from the OECD 422 study with aluminium, benzoate C16-18 fatty acid complexes are read across to the registered substance. There were no indications of any reproductive toxicity when administered to rats on this study so it can be concluded that no classification for this endpoint is required.

Annex IX requires the consideration of a further general fertility and reproductive toxicity study if the 28-day study indicates adverse effects on reproductive organs or tissues. Since no such effects on the male or female reproductive system were seen in the key OECD 422 study on the supporting substance aluminium, benzoate C16-18 fatty acid complexes, the conduct of a further reproductive study onthe registered substancehas been waived.

The registered substance is manufactured and used solely in an inert carrier. The substance and aluminium, benzoate, C16-18 fatty acids complexes are used specifically as thickeners in industrial greases and lubricants and, as such, typically occur in situ in base oil. The interactions between the thickener and base oil do not strictly fall under the definitions of a reaction product nor do they act as a simple mixture of components due to matrix effects which occur in the manufacturing/ blending process. Matrix effects should be taken into account when assessing whether adverse effects would be seen in grease products because entrainment of grease thickeners during the manufacturing process as part of the grease matrix severely limits exposure and will have a significant impact on the outcome of any risk assessments. The fact that matrix effects do occur is a point recognised in the OECD Lubricant Emission Scenario document (2004) and it is therefore reasonable to assess the influence the process of manufacturing the thickener in an inert base oil has on factors such as availability, which are critical to assessing the potential risks posed by the grease thickeners when in the grease.

Leaching tests have been undertaken to assess whether the metallic components of the metal-soap and metal-complex-soap grease thickeners remain in base grease(s) and hence determine the degree of availability of the thickeners (see section 4.5 and Appendix 2 of the CSR). The results of the study show no leaching of the thickener from the base oil and a lack of bioavailability of the substance in aqueous media. The same lack of bioavailability is expected in GI fluids as well and as a further demonstration of this lack of availability, it is proposed to conduct leaching studies on aluminium thickeners in base oil using fed state simulated intestinal fluid (FeSSIF). This approach provides sufficient information to assess the risks associated with the aluminium thickener complexes following exposure of mammals by the oral route. Also, the use of inestinal fluid has been used to evaluate the solubility of drugs for the prediction of oral absorption (Augustijns 2014).


Justification for selection of Effect on fertility via oral route:
This screening study provides relevant read-across experimental data on this endpoint from a related substance, aluminum, benzoate C16-18 fatty acid complexes in which a clear NOAEL is identified.

Effects on developmental toxicity

Description of key information
Data are taken from a read-across oral reprotoxicity screening study in rats with aluminum, benzoate C16-18 fatty acid complexes, and conducted according to OECD 422, in which no adverse effect was seen in any of the developmental parameters examined at any dose.
Link to relevant study records

Referenceopen allclose all

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
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
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
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
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
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
Details on maternal toxic effects:
Maternal toxic effects:no effects
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
Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects: No effects
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.
Key result
Developmental effects observed:
no
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.

Endpoint:
developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and there is no or no significant human exposure
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
A GLP Regulatory study (OECD 422) was conducted in rats by daily oral gavage administration to screen for potential effects of aluminum, benzoate C16 -18 fatty acid complexes on reproduction and offspring development and the results are read across to the registered substance. There were no effects on any of the reproductive or developmental parameters measured. The limitations of this screening test preclude the conduct of further testing on the F1 developmental parameters.

The OECD 422 study was conducted on the aluminum, benzoate C16 -18 fatty acid complexes manufactured in situ in a medicinal grade white oil base and the maximum dose that could be administered was limited by the physico-chemical characteristics of the dosing preparation. Because of the physico-chemical constraints, the limit dose of actual aluminium, benzoate C16-18 fatty acid complexes could not be attained. Since no general toxicity or developmental or fertility effects were observed in the OECD 422 study and the maximum dose was below the limit dose of 1500 mg/kg/day (actual maximum dose achieved was 225 mg/kg bw/day, which was considered to be the NOAEL), then the likelihood of identifying any further developmental toxicity responses at the same concentrations is extremely low.

This lack of any systemic toxicity was also observed in the acute oral and dermal toxicity studies, which were conducted on aluminium, benzoate, C16-18 fatty acid complexes isolated from an oil base and re-dissolved in arachis oil, at doses up to 2000 mg/kg bw by either route and an acute oral toxicity study, which was conducted on the registered substance as a suspension in poly alpha olefin, at doses up to 2000 mg/kg bw.

It was previously proposed to conduct a Regulatory OECD 414 study in rats with aluminum, benzoate C16 -18 fatty acid complexes and read across these results to the registered substance. However, further review of the available data has indicated that this testing is not required and it is proposed instead to provide weight of evidence to show a lack of bioaccessibility for the registered substance and the structural analogue aluminium benzoate C16-18 fatty acids complexes.

The registered substance is manufactured and used solely in situ in an inert carrier. The registered substance and aluminium, benzoate, C16-18 fatty acids complexes are used specifically as thickeners in industrial greases and lubricants and as such typically occur in base oil. The interactions between the thickener and base oil do not strictly fall under the definitions of a reaction product nor do they act as a simple mixture of components due to the matrix effects which occur in the manufacturing/blending process. Matrix effects should be taken into account when assessing whether adverse effects would be seen in grease products because entrainment of grease thickeners during the manufacturing process as part of the grease matrix severely limits exposure and will have a significant impact on the outcome of any risk assessments. The fact that matrix effects do occur is a point recognised in the OECD Lubricant Emission Scenario document (2004) and it is therefore reasonable to assess the influence the process of manufacturing the thickener in an inert base oil has on factors such as availability, which are critical to assessing the potential risks posed by the grease thickeners when in the grease.

Leaching tests have been undertaken to assess whether the metallic components of the metal-soap and metal-complex-soap grease thickeners remain in base grease(s) and hence determine the degree of accessibility of the thickeners (see section 4.6 and Appendix 2 of the CSR). The results of the study show no leaching of the thickener from the base oil and a lack of bioaccessibility of the substance in aqueous media. The same lack of bioaccessibility is expected in GI fluids as well. It is considered that both the registered substance and aluminium, benzoate, C16-18 fatty acids complexes in an oil base are not considered to be bioaccessible. As a further demonstration of this lack of accessibility, it is proposed to conduct a leaching study in fed state simulated intestinal fluid (FeSSIF) using the registered substance and the structural analogue aluminium, benzoate, C16-18 fatty acids complexes. This approach provides sufficient information to assess the risks associated with the aluminium complex thickeners, and by replacing the proposed vertebrate test with a weight of evidence argument, also follows the principle of avoiding unnecessary animal testing.

This substance has been registered by a Member of the European REACH Grease Thickeners Consortium (ERGTC). A number of decisions have been made in the dossier with regard to the approach taken for registering the substance including the testing strategy and the justification for waiving certain endpoints. Several of the decisions reflect the technical difficulties of testing the substance and the relevance of data with regard to the potential for exposure, given that the substance typically occurs in situ in base oil. A face to face meeting between the ERGTC and ECHA was held in Helsinki on 8th September 2016 which discussed many of these topics and a copy of the minutes from the meeting are attached to the dossier (See section 13 of IUCLID). Therefore, if there are any queries or concerns which arise when the dossier is reviewed, it is requested that the reviewer discuss these with the ERGTC (ERGTC@wca-consulting.com) as there may be background information and previously discussions between the ERGTC and ECHA available which are relevant.
Abnormalities:
not specified
Developmental effects observed:
not specified
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Read across data
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

In accordance with the Regulation (EC) No 1907/2006, Annex XI, section 1.5, read-across to aluminum, benzoate C16-18-fatty acids complexes has been used to fulfil REACH information requirements where appropriate and is justified by the chemical structures and common physiological active moieties of the substances. The chemical structures of the target and read-across substances are very closely aligned. The aluminium cation, a long chain fatty acid, and the –Al=O (-AlOH in aqueous solution) moieties are identical in both substances. The key difference is that read-across substance contains a benzoate moiety linked to the aluminium cation, which is absent from the target substance. Benzoic acid and benzoates have been well characterized (eco)toxicologically, but in this case generating experimental data on the aluminium salt containing benzoate would be expected to demonstrate a ‘worst case’ hazard profile when compared to the target substance. Since no intrinsic toxicity could be demonstrated from any of the Annex VII or VIII endpoints with the benzoate-containing aluminium salt, then these results can be read across to the target substance without restriction.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical: Aluminum, benzoate, C16-18 fatty acids complexes (EC: 303-385-6, CAS: 94166-87-7)

See robust study summaries for further details on the identity of the tested substances and IUCLID dataset for further information on the substance identity and the data to support the read across justification.


3. ANALOGUE APPROACH JUSTIFICATION
Aluminum, benzoate C16-18-fatty acids complexes is considered suitable for read-across as it contains a fatty acid moiety coordinated to an aluminium atom. The chemical structures of the target and read-across substances are very closely aligned; both substances consist of aluminium salts of fatty acids. The aluminium cation, a long chain fatty acid, and the –Al=O (-AlOH in aqueous solution) moieties are identical in both substances.

The fatty acids present in both substances are the same, consisting of a mixture of C16 and C18 chain lengths at approximately a 1:2 ratio. The C16 and C18 fatty acid moieties are derived from natural fatty materials, or substances which are chemically indistinguishable from natural fatty acids. The fatty acid moieties are considered not to be hazardous to humans as they are natural constituents of the human body and essential components of a balanced human nutrition. REACH Annex V, Entry 9, groups fatty acids and their potassium, sodium, calcium and magnesium salts, including C6 to C24, predominantly even-numbered, unbranched, saturated or unsaturated aliphatic monocarboxylic acids. Provided that they are obtained from natural sources and are not chemically modified, the substances included in REACH Annex V, Entry 9 are exempt from registration, unless they are classified as dangerous (except for flammability, skin irritation or eye irritation) or they meet the criteria for PBT/vPvB substances. The fatty acid components of the two substances are therefore expected to be exempt under REACH.

Fatty acids are an endogenous part of every living cell and are an essential dietary requirement. They are absorbed, digested and transported in animals and humans. When taken up by tissues they can either be stored as triglycerides or can be oxidised via the ß-oxidation and tricarboxylic acid pathways. The ß-oxidation uses a mitochondrial enzyme complex for a series of oxidation and hydration reactions, resulting in a cleavage of acetate groups as acetyl CoA. Acetyl CoA is used mainly to provide energy but also to provide precursors for numerous biochemical reactions. Alternative minor oxidation pathways can be found in the liver and kidney (ω-oxidation and ω-1 oxidation) and in peroxisomes for ß-methyl branched fatty acids (α-oxidation). The metabolic products can then be incorporated for example into membrane phospholipids.

Comparison of the data for the two substances indicates that they are expected to have similar properties. Neither the target or read-across substance meets the criteria for classification for physico-chemical, environmental or human health endpoints, based on the available data.

On the basis of the physico-chemical results, the substances are not flammable and have similar densities. The low vapour pressure results indicates that hazards associated with the atmospheric compartment or inhalation routes of toxicity are not expected to be relevant. The substances show similar water solubility, without surface active properties, indicating that they are likely to have similar behaviour in the aquatic environment.

Although the read-across substance met the criteria for ready biodegradability and the target substance (tested as a 50% concentration in pharmaceutical white oil) did not, neither substance was inhibitory to micro-organisms at the concentration tested. The difference in biodegradation results is expected to derive from the presence of the base oil in the target substance sample, which is designed to minimise leaching of the grease thickener, and therefore less of the grease thickener would have been available for degradation by the micro-organisms.

There are no results available for the ecotoxicity of the target substance and therefore comparison of the effect concentrations against the read-across substance is not possible. However, leaching studies on grease thickeners in base oils have been used to assess the potential bioavailability of the grease components. The bioavailability potential of the water accommodated fractions (WAFs) of metal (lithium and calcium) soap complex based grease thickeners was assessed using a solid-phase micro-extraction (SPME) method combined with gas chromatography (GC). This approach was complemented with metal ion analysis to determine whether the metal leaches out of the base grease during WAF preparation and the ecotoxicity of WAFs was also monitored using an in vitro Microtox assay. The SPME-GC data confirmed that there was negligible leaching of the thickeners from base oils in the samples tested, with measurements for calcium and lithium below the limit of detection (<0.1 mg/L) and the screening ecotoxicity data also showed a lack of toxicity of the greases.

The results of the bioavailability potential of the WAFs, the metal ion analysis and the screening ecotoxicity of lithium and calcium based complexes have been read across to aluminium based thickeners. All of these metal salts of fatty acids are expected to behave in a very similar manner when entrained within a grease matrix, with high temperature stability indicating that the thickener structure is robust and resistant to diffusion out of the oil. Dissolution of grease thickeners from grease into water is very unlikely as the thickeners are poorly water soluble and the thickeners are embedded in the hydrophobic grease matrix and thus unlikely to leach out. Therefore, although there are no data on the ecotoxicity of the target substance, no effects are expected based on the lack of bioavailability of the thickener.

These data on the potential for leaching of other metal salt complex based grease thickeners have been read across to both the target and read across substances. On the basis of these results, it is expected that neither the target nor the read across substance would leach from the base oil in which they are typically marketed and therefore neither substance would be bioavailable. Thus, reading across data from the source substance tested in its isolated form is considered robust as it provides a worst-case conclusion for the target substance which is only manufactured in an inert carrier, typically base oil. In order to provide further evidence for the lack of bioavailability, it is proposed to undertake leaching studies on the target and read-across substances themselves. Dependent on the results, the two studies would then be used to show the similarity in the bioavailability of the two substances and provide further weight of evidence for the read-across approach.

The available mammalian toxicity data show that neither the target nor read-across substance would be classified as irritating to skin or eyes and would not be classified for acute oral toxicity, with LD50 values of >2000 mg/kg. Although no other data are available for comparison of the potential mammalian toxicity of the two substances, the target and read-across substances are expected to behave in a very similar manner. As grease thickeners are entrained within grease matrices which are robust and resistant to diffusion out of the oil, neither substance is expected to be bioavailable. In order to provide further evidence for the lack of bioavailability, it is proposed to undertake leaching studies in fed state simulated intestinal fluid (FeSSIF) on the target and read-across substances. Dependent on the results, the two studies would then be used to show the similarity in the bioavailability of the two substances and provide further weight of evidence for the read-across approach.

For the developmental toxicity of the substances, read across from the source to the target substance is considered justified. Both substances would not leach when in situ in base oil during use as grease thickeners and are not expected to be bioavailable. The substances would dissociate into inorganic aluminium species and fatty acids (plus benzoic acid for the source substance), the organic components of which are readily metabolised. As the fatty acid components are essential nutrients to many organisms and are not expected to be hazardous (and the benzoate component of the source substance is not expected to be hazardous), the toxicity is expected to be driven by the aluminium component, so would be the same in both the source and target substances. As such, read across from the source substance is considered to provide a worst-case scenario for the target substance.

4. DATA

T = target substance (tests were undertaken on a sample prepared as a 50% w.w. concentration in medicinal white oil unless otherwise indicated)
RA = read-across substance

- State: Liquid (T), Solid (RA)
- Melting point: 21°C (T), 224°C (RA)
- Relative density: 0.933 (T), 1.08 (RA)
- Vapour pressure: 0.00015 Pa (T), 0.000044 Pa (RA)
- Surface tension: 72.5 mN/m (T), 72.6 mN/m (RA)
- Water solubility: ≤0.00015 g/L (T), ≤0.00026 g/L (RA)
- Flash-point: 159°C (T), No data available for RA
- Flammability: No data available for T, Not flammable (RA)
- Self-ignition temperature: 374°C (T), 383°C (RA)
- Viscosity: 174.3 mm2/s at 100°C (T), No data available for RA
- Biodegradation: Not readily biodegradable (31%) (T), Readily biodegradable (79%) (RA)
- Acute aquatic invertebrates: No data available for T, EL50 (48 h): > 100 mg/L (RA)
- Algae: No data available for T, EL50 (72 h): > 100 mg/L and NOELR (72 h): 100 mg/L (RA)
- Aquatic microorganisms: NOEC (28 d): 6.7 mg/L (T), NOEC (28 d): 15.4 mg/L (RA)
- Acute fish: No data available for T, LL50 (96 h): > 100 mg/L (RA)
- Skin irritation: Not irritating (T), Not irritating (RA)
- Eye irritation: Not classified (T), Not classified (RA)
- Skin sensitisation: No data available for T, Not sensitising (RA)
- In vitro gene mutation in bacteria: No data available for T, Negative (RA)
- Acute toxicity, oral route: LD50: > 2000 mg/kg (T, test undertaken on solid (isolated) form of the substance), LD50 >2000 mg/kg (RA)
- Acute toxicity, dermal route: No data available for T, LD50 >2000 mg/kg (RA)
- In vitro cytogenicity: No data available for T, Negative (RA)
Reason / purpose:
read-across source
Details on maternal toxic effects:
- Maternal toxic effects: No effects
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
Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects: No effects
Key result
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.
Key result
Developmental effects observed:
no
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:

Aluminium, benzoate, C16 -18 fatty acids complexes is considered suitable for read across as it contains a fatty acid moiety coordinated to an aluminium atom. Although it contains a coordinated benzoate ion, no toxicological effects were observed and therefore it is concluded that the benzoate ion does not contribute any additional toxicity to the substance. Aluminium, benzoate, C16 -18 fatty acids complexes showed no toxicological effects in an OECD 422 study (Harlan 2013). Therefore, aluminium benzoate C16 -18 fatty acids complexes is considered to have a NOAEL of 1500 mg/kg bw/day (225 mg/kg bw/day active ingredient) and this has been read across to the target substance.

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
225 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Guideline study conducted to GLP
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A key subacute toxicity and reproductive toxicity screen, using the OECD 422 study design, was conducted in rats on aluminum, benzoate C16-18 fatty acid complexes via oral gavage administration. The test material was administered at dose levels of 0, 375, 750 and 1500 mg/kg bw/day nominal, equating to 56.3, 113 and 225 mg/kg bw/day Active Ingredient. There were no treatment-related effects at any dose level on any of the reproductive parameters evaluated in this study, or in any of the developmental parameters evaluated. Based on these data, the NOAEL for reproductive and developmental toxicity was 1500 mg/kg bw/day, equivalent to 225 mg/kg bw/day Active Ingredient.

Data taken from the OECD 422 study with aluminium, benzoate C16-18 fatty acid complexes are read across to the registered substance. There were no indications of any developmental toxicity when administered to rats on this study so it can be concluded that no classification for this endpoint is required.

Annex IX requires the consideration of a further general fertility and reproductive toxicity study if the 28-day study indicates adverse effects on reproductive organs or tissues. There were no effects on any of the reproductive or developmental parameters measured. The limitations of this screening test preclude the conduct of further testing on the F1 developmental parameters. It was previously proposed to conduct a Regulatory OECD 414 study in rats with aluminum, benzoate C16 -18 fatty acid complexes and read across these results to the registered substance. However, further review of the available data has indicated that this testing is not required and it is proposed instead to conduct leaching studies on aluminium thickeners in base oil using fed state simulated intestinal fluid (FeSSIF). This approach provides sufficient information to assess the risks associated with the aluminium thickener complexes following exposure of mammals by the oral route. Also, the use of inestinal fluid has been used to evaluate the solubility of drugs for the prediction of oral absorption (Augustijns 2014).

The registered substance is manufactured and used solely in situ in an inert carrier. The substance and aluminium, benzoate, C16-18 fatty acids complexes are used specifically as thickeners in industrial greases and lubricants and as such typically occur in situ in base oil. The interactions between the thickener and base oil do not strictly fall under the definitions of a reaction product nor do they act as a simple mixture of components due to the matrix effects which occur in the manufacturing/ blending process. Matrix effects should be taken into account when assessing whether adverse effects would be seen in grease products because entrainment of grease thickeners during the manufacturing process as part of the grease matrix severely limits exposure and will have a significant impact on the outcome of any risk assessments. The fact that matrix effects do occur is a point recognised in the OECD Lubricant Emission Scenario document (2004) and it is therefore reasonable to assess the influence the process of manufacturing the thickener in an inert base oil has on factors such as availability, which are critical to assessing the potential risks posed by the grease thickeners when in the grease.

Leaching tests have been undertaken to assess whether the metallic components of the metal-soap and metal-complex-soap grease thickeners remain in base grease(s) and hence determine the degree of availability of the thickeners (see section 4.6 and Appendix 2 of the CSR). The results of the study show no leaching of the thickener from the base oil and a lack of bioavailability of the substance in aqueous media. The same lack of bioavailability is expected in GI fluids as well and as a further demonstration of this lack of availability, it is proposed to conduct leaching studies on aluminium thickeners in base oil using fed state simulated intestinal fluid (FeSSIF). This approach provides sufficient information to assess the risks associated with the aluminium thickener complexes following exposure of mammals by the oral route. Also, the use of intestinal fluid has been used to evaluate the solubility of drugs for the prediction of oral absorption (Augustijns 2014).

Justification for selection of Effect on developmental toxicity: via oral route

This screening study provides relevant read-across experimental data on this endpoint from a related substance, aluminum, benzoate C16-18 fatty acid complexes in which a clear NOAEL is identified.

Justification for classification or non-classification

The substance is not classified for reproductive toxicity. No adverse reproductive or developmental toxicity effects were observed in a relevant study conducted on the supporting substance aluminum, benzoate C16-18 fatty acid complexes.