Lanthanum fluoride

Endpoint summary

Description of key information

Oral (28-day): Male/female NOAEL 1000 mg/kg bw, female; OECD 422, Charles River (2013)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 October 2012 to 24 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8-10 weeks (treatment initiation).
- Weight at study initiation: 301 - 369 g (males); 232 - 285 g (females) at treatment initiation.
- Housing: Animals were housed in polycarbonate cages with stainless steel grid tops and solid bottoms, with approximate dimensions of 61 x 43.5 x 24 cm. The animals were initially housed 2 or 3 per cage. A few days prior to pairing for mating, males were transferred to individual cages with a stainless steel grid insert. After mating, the males were re-housed with their original cage mates. Mated females were transferred to individual solid bottom cages. White paper tissue was supplied as nesting material from Day 20 of gestation.
- Diet: ad libitum
- Water: tap water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 22 °C
- Humidity (%): 32 - 83 %
- Air changes (per hr): 10 air changes per hour (minimum)
- Photoperiod (hrs dark / hrs light): a 12 hour light/dark cycle was maintained
- Environmental enrichment: Chewing objects and hiding devices were provided to the animals as appropriate for psychological/environmental enrichment.

IN-LIFE DATES: From 29 October 2012 to 17 December 2012

Route of administration:

oral: gavage

Vehicle:

other: 1% (w/v) Carboxymethylcellulose (high viscosity) in Milli-Q water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
- The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4 °C, and dispensed daily. They were prepared by adding an appropriate amount of vehicle to the required amount of test material and were mixed by a high sheer mixer and magnetic stirring until a visibly homogenous formulation was obtained. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes prior to dosing and continuously during dosing. All formulations were used within the 8-day stability period that had been previously established.

- Dose volume: 10 mL per kg body weight.

- The volume administered to each animal was determined on each day by the weight of that animal as measured at the time of administration, except during late gestation; from Day 16 of gestation until when parturition was complete, the dose volume was determined by the weight of the animal on Day 16 of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

From each formulation, duplicate sets of top, middle and bottom samples were taken at each sampling time point and sent to the analytical laboratory for analysis (duplicate middle samples only obtained from Control formulations). Additional triplicate samples from the top, middle and bottom (middle sample only from control) were also taken as back-up samples. The results of the sample concentration analyses were considered acceptable if all results were within ± 15 % of theoretical concentration. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentration of ≤ 10 % for each group.

- Preparation of test solutions: the appropriate amount of formulation was weighed into a digestion vessel and digested in aproximately 10 mL of 69 % nitric acid. Once digested, the samples were transferred to a 50 mL volumetric flask with washings made to volume in 2 % nitric acid. The samples were further diluted in 2 % nitric acid to give sample solution concentrations within the range of the calibration standard used. Samples and standards were analysed by ICP-OES.

- Preparation of calibration standards: 20 mg of test material was added to a digestion vessel and digested in approximately 10 mL of 69 % nitric acid. Once digested the solution was transferred to a 100 mL volumetric flask with washings and made to volume in 2 % nitric acid to obtain a stock solution with a concentration of 0.200 mg/mL lanthanum fluoride. The stock solution was diluted volumetrically in 2 % nitric acid to obtain target concentrations of approximately 0.00100, and 0.0100 mg/mL lanthanum fluoride.

- ICP-OES conditions:
> Instrument: Perkin Elmer Optima 8000 ICP-OES
> Elements and wavelength: La (398.852 nm)
> No. of replicates: 3
> Delay time: 60 seconds
> Read time: set to auto
> Minimum read time: 1 second
> Maximum read time: 5 seconds
> Points per peak: 7
> Plasma conditions:
Plasma: 8 L/min
Auxiliary: 0.2 L/min
Nebuliser: 0.7 L/min
RF power: 1300 Watts
Plasma viewing mode: radial
> Peristatic pump sample flow rate: 1.50 mL/min
> Wash:
Frequency: between samples
Flow rate: 1.50 mL/min
Normal time: 30 seconds
Wash solvent: Milli-Q water
> Diluent: 69 % nitric acid (for digestion); 2 % nitric acid (for dilutions)
> Microwave digestion:
Ramp: 20 min to 200 °C
Hold: 20 min at 200 °C
Cool down: 45 min

- Calculations: a calibration curve was constructed by plotting the mean peak areas of the standards (including the calibration blank) against the relevant lanthanum fluoride concentration in mg/mL. The concentration of lanthanum fluoride in the test samples was determined by interpolation of this line.

Concentration of lanthanum fluoride in formulations (mg/mL) = (C x D x P) / W

Where
C = concentration of the sample solution obtained by interpolation from the calibration line (mg/mL)
D = dilution factor
P = density of the formulation (g/mL)
W = weight of aliquot (g)

- The limit of detection (LOD) estimated from an analyte response at the level of 3.3 times the ratio of the standard deviation of the blank to the slope of the calibration curve, was estimated to be 1.59 x10^-6 mg/mL Lanthanum Fluoride.
- The limit of quantification (LOQ) estimated from the an analyte response at the level of 10 times the ratio of the standard deviation of the blank to the slope of the calibration curve, was estimated to be 4.81 x10^-6 mg/mL Lanthanum Fluoride.

Duration of treatment / exposure:

The males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (ca 4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca 6 weeks of treatment).
* Animals 41 (Group 1 female) and 59 (Group 2 female) were not dosed on their respective days of parturition due to the animals starting parturition prior to the commencement of dosing that day.

Frequency of treatment:

Animals were dosed once daily.

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
other: nominal conc.

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected after a review of existing relevant toxicological data. Including a separate 14-day dose range finding study, where dose levels up to 1000 mg/kg/day produced no adverse reaction to treatment.
- Animal assignment: On arrival from the suppliers, the animals were housed in cages. The cages were suspended on a series of racks. Male and female cages were racked separately. Cages were allocated to treatment group by the use of randomly sequenced numbers in such a way that each complete rack contained representatives from all treatment groups. During pre-trial Animals 45 (Group 1 female) and 77 (Group 4 female) were replaced with spare Animals 83 and 84, respectively, due to findings in the pre-trial ophthalmic examinations. Animals 45 and 77 were not used and were therefore considered not to be part of this study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked for viability early morning and as late as possible each day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week starting in pretrial, all animals received a detailed clinical examination, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded one week prior to the start of treatment. From the start of treatment, the individual body weights were recorded daily.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was measured for both sexes weekly, starting 1 week prior to dosing until pairing for mating.
After pairing, the female food consumption was measured over Days 0-7, 7-14 and 14-20 of gestation and Days 0-4 of lactation. Male food consumption did not recommence after pairing for mating.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretrial, week -1 (all animals); week 4 (all males); shortly prior to sacrifice (all females).
- The eyes were examined using an indirect ophthalmoscope after the application of a mydriatic agent (1 % Tropicamide, Mydriacyl®). The following areas were evaluated: anterior, lenticular and fundic areas.

HAEMATOLOGY: Yes. Samples for haematology were collected via the tail vein. Blood was then transferred into plastic tubes containing anticoagulant.
- Time schedule for collection of blood: week 4 (males); ca day 4 of lactation (females).
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: first 5 animals per group (males); first 5 animals per group which reared their litter to day 3 of lactation (females).
- Approximately 0.5 mL of blood was taken into tubes containing EDTA and analysed for the following: Red blood cell count, Haemoglobin concentration, Haematocrit, Mean corpuscular volume, Red blood cell distribution width, Mean corpuscular haemoglobin concentration, Mean corpuscular haemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unclassified cells, Other cells (as appropriate).

CLINICAL CHEMISTRY: Yes. Samples for clinical chemistry were collected via the tail vein. Blood was then transferred into plastic tubes containing anticoagulant.
- Time schedule for collection of blood: week 4 (males); ca day 4 of lactation (females)
- Animals fasted: No
- How many animals: first 5 animals per group (males); first 5 animals per group which reared their litter to day 3 of lactation (females)
- Blood samples (1.0 mL) were taken into tubes containing lithium heparin which were then centrifuged and the plasma was analysed for the following: Urea, Glucose, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatise, Creatine phosphokinase, Lactate dehydrogenase, Sodium, Potassium, Chloride, Total protein, Albumin, Globulin, Albumin/globulin ratio, Cholesterol, Creatinine, Total bilirubin, Calcium, Phosphate.

DETAILED FUNCTIONAL OBSERVATIONS
- Time schedule: weekly from pre-treatment (week -1)
- Cageside observations included: Prostration, Lethargy, Writhing, Circling, Breathing abnormalities, Gait abnormalities, Tremor, Fasciculation, Convulsions, Biting (of cage components or self mutilating), Vocalisations, Piloerection and Ease of removal from the cage.
Body temperature was taken directly from an implanted electronic chip and recorded.
Condition of the eyes, checked for:
Pupillary function
Miosis
Mydriasis
Exophthalmos
Encrustation
Lacrimation.
Condition of the coat, presence of salivation and overall ease of handling were also assessed.

- Observations in a standardized area (2 min observation):
Latency (time to first locomotory movement)
Level of mobility
Rearing
Grooming
Urination/defecation
Arousal (level of alertness)
Posture, tremor/convulsions, vocalisation, piloerection – recorded as for cageside observations
Palpebral closure
Gait abnormalities
Stereotypy (excessive repetition of behaviours) and/or unusual behaviours.

- Functional Tests (Full Examination)
The following additional functional assessments were performed on 5 males per group during week 4 and on 5 females per group during lactation (the first 5 females per group to rear their litter to day 3 of lactation). These assessments were performed at an approximately standardised time of day and prior to blood sampling.
> Reaction to sudden sound (click above the head).
> Reaction to touch on the rump with a blunt probe.

> Grip strength:
A strain gauge was used, to which is attached a wire pull-mesh. Once the animal had gripped the mesh, the body was pulled until its grasp was broken; the strain gauge recorded the force required. The procedure was repeated 3 times for the forelimbs and 3 times for the hindlimbs, and the mean fore and hind grip strengths calculated.

> Pain perception:
This was assessed by measurement of the tail flick response. The apparatus used shone a calibrated infra-red heat source onto the tail and automatically measured the reaction time of the animal (accurate to 0.1 s). It was ensured that no visible injury to the tail was caused during this test.

> Landing Foot Splay:
Corn oil was applied to the hind paws of each animal. The animal was then held in a horizontal, prone position with the nose ca 30 cm above a bench surface covered with absorbent paper. When the animal was calm, it was dropped. The distance between the prints of the central footpads was measured and the average measurement recorded. The procedure was repeated 3 times.

> Motor activity:
Each animal was placed in an individual monitoring cage, scanned by a motion sensor utilising infra-red pyroelectric detectors. Movement was detected in 3 dimensions anywhere in the cage, and was differentiated into large and small movements. Each animal was monitored for one session of 1 h for males and 0.5 h for females. Activity counts were recorded over successive periods of 5 min each.

> Other physical/functional abnormalities:
Any other abnormality not already recorded in the above screening battery.

COAGULATION
Samples for coagulation were collected via the tail vein. Blood was then transferred into plastic tubes containing anticoagulant.
- Time schedule for collection of blood: week 4 (males); ca day 4 of lactation (females)
- Animals fasted: No
- How many animals: first 5 animals per group (males); first 5 animals per group which reared their litter to day 3 of lactation (females)
- Blood samples (0.9 mL) were taken into tubes containing 0.1 mL trisodium citrate (3.8% (w/v)). The final sample volume was as close as possible to 1.0 mL to give a final concentration of 0.38 % (blood to citrate ratio of 9:1). The citrated blood samples were centrifuged and the plasma separated into plastic tubes and analysed for the following: Activated partial thromboplastin time, Fibrinogen, Prothrombin time.

Sacrifice and pathology:

The males were killed when mating was completed and the animals had been dosed for at least 4 weeks (Study Day 30).
The females and litters were killed between Day 5 and 7 of lactation (Study Days 43, 46 - 47 and 50).
Animals 10 days of age or more were killed by exposure to carbon dioxide followed by exsanguination. Animals less than 10 days of age were killed by intra-peritoneal injection of sodium pentobarbitone.

GROSS PATHOLOGY: Yes
All adult animals surviving to scheduled euthanasia were subjected to a complete necropsy examination which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

ORGAN WEIGHTS: Yes
The following organs were weighed at necropsy for all adult animals surviving to terminal kill: Brain, Epididymides, Adrenal glands, Pituitary gland, Prostate gland, Thyroid glands, Heart, Kidneys, Liver, Lung, Ovaries, Spleen, Testes, Thymus, Uterus.

HISTOPATHOLOGY: Yes
Representative samples of the following tissues were collected from all adult animals and preserved in 10 % neutral buffered formalin, unless otherwise indicated:
Aorta, Blood smear* (Animals sacrificed prematurely only), Bone marrow smear# , Bone marrow (femur, sternum), Femur, Rib, Sternum, Brain, Cervix, Epididymides, Eyes~, Adrenals, Harderian glands~ , Lacrimal glands, Mammary gland, Parathyroid glands, Pituitary gland, Prostate gland, Salivary glands, Seminal vesicle, Thyroids, Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidneys, Large intestine (caecum, colon, rectum), Larynx, Liver, Lung, Mandibular lymph node, Mesenteric lymph node, Skeletal muscle, Nasal cavity, Optic nerve~, Sciatic nerve, Oesophagus, Ovaries, Oviducts, Pancreas, Pharynx, Skin, Small intestine (duodenum, ileum, jejunum), Spinal cord, Spleen, Stomach, Testes+, Thymus, Tongue, Trachea, Ureter x2, Urinary bladder, Uterus, Vagina.
* Air dried
# Fixed in methanol and then air dried
~ Preserved in Davidson’s fixative
+ Preserved in Modified Davidson’s fixative

- Bone Marrow Smears
Duplicate bone marrow smears were taken at necropsy from all adult animals. Both bone marrow smears were stained using May-Grunwald-Giemsa as the Romanowsky stain. Bone marrow smears were not evaluated.

- Histopathology
The tissues, as listed above (except animal identification, nasal cavity, blood smears and bone marrow smears), were processed to paraffin wax block from selected animals. The testes of all male animals were processed to wax impregnation. Sections were cut 4-6 µm thick, stained with haematoxylin and eosin from 5 males and 5 females in the Control and High dose groups (the same animals that were used for laboratory investigations). An additional PAS-Haematoxylin stained slide was prepared from the testes and epididymides of the selected males.

Statistics:

Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group.
Selected body weight and food consumption data and all haematology, coagulation, clinical chemistry and selected functional observational battery and motor activity data was analysed for homogeneity of variance using the ‘F Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test ie pairwise comparisons were made only if the overall F test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.

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:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

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

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no premature decedents on this study. Treatment at levels up to 1000 mg/kg/day in males and females produced no clinical observations that were considered related to treatment with the test material. In both sexes and in all dose groups there was high incidence of soft faeces observed. As this was present in almost all Control animals, this was considered to be an effect caused by the vehicle and not related to treatment with the test material.

BODY WEIGHT AND WEIGHT GAIN
In the treated males the group mean body weight gains appeared to be slightly reduced when compared to Controls. The high standard deviation in Controls shows a high degree of variation between the individual animals within this group. The group mean gain has also been skewed by 2 outlier males who had very high body weight gains throughout the duration of the study (Animals 2 and 10), when compared to the other males in this group. Therefore this apparent difference in body weight gain is considered incidental and not related to treatment with the test material. At levels up to 1000 mg/kg/day the group mean body weight gain for females prior to mating and throughout gestation and lactation were similar to Control.

FOOD CONSUMPTION
Group mean food consumption prior to pairing for mating was similar to Controls in both males and females. The group mean food consumption in all treated females was similar to Controls during gestation and lactation.

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmoscopy findings which were considered to be related to treatment with the test material. All findings were considered to be typical of rats of the age and strain used.

HAEMATOLOGY
In both sexes all haematology and coagulation parameters were similar to Controls. At 1000 mg/kg/day there was a slight reduction in white blood cell counts in females, when compared to Controls. Only 2/5 females were found to have values slightly below the range of the Control females. These slight intergroup differences were considered incidental and not related to treatment with the test material.

CLINICAL CHEMISTRY
In males and females dosed at 300 mg/kg/day and above there were slight differences in lactate dehydrogenase, when compared to Controls. At 300 mg/kg/day the group mean lactate dehydrogenase was reduced by 30 % in males and 21 % in females and at 1000 mg/kg/day the group mean lactate dehydrogenase was reduced by 28 % in males and 25 % in females, when compared to Controls. There was also an increase in the number of animals
affected throughout the treated groups, rising from 3/5 males dosed at 300 mg/kg/day to 4/5 males dosed at 1000 mg/kg/day. Similarly in females, 2/5 females dosed at 300 mg/kg/day were affected compared to 3/5 females dosed at 1000 mg/kg/day.
In males there was a dose-related slight decrease in group mean creatine phosphokinase in all treated groups, when compared to Controls (18 % reduction at 100 mg/kg/day, 20 % reduction at 300 mg/kg/day and 23 % reduction at 1000 mg/kg/day). At 300 mg/kg/day and above in females there was a slight decrease in group mean creatine phosphokinase, reduced by 13% at 300 mg/kg/day and 15 % at 1000 mg/kg/day when compared to Controls.
The differences in LDH and CPK never achieved statistical significance however due to the increase in the number of animals affected throughout the treated groups (LDH only) and due to the similar pattern of effect in males and females it cannot be discounted that these differences could be related to treatment with the test material.
All other clinical chemistry parameters in males and females were similar to Controls. Any slight intergroup differences were considered incidental and too small to be attributed to treatment with the test material.

NEUROBEHAVIOUR
The type and distribution of neurotoxicity clinical observations in both males and females did not indicate any effect of treatment. In all dose groups, all detailed functional observations in males and females were comparable to Controls for the duration of the study. Any slight intergroup differences were considered too small to be attributed to treatment. Slight inter-group differences in motor activity were intermittent and considered incidental, therefore could not be associated with treatment with the test material.

ORGAN WEIGHTS
In all treated animals the organ weights were similar to Controls. Any slight intergroup differences in the organ weights were too small to be related to administration of the test material.

GROSS PATHOLOGY
No test material-related gross findings were noted. The gross findings observed were considered incidental and of the nature commonly observed in this strain and age of rat.

HISTOPATHOLOGY
There were no treatment-related microscopic findings. All of the histopathological findings encountered were considered to have arisen spontaneously or at post mortem. The incidence and severity of microscopic findings were similar in control and treated animals and, therefore, were considered unrelated to administration of the test material.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical chemistry

Critical effects observed:

not specified

Group 1: Control group

Group 2: 100 mg/kg group

Group 3: 300 mg/kg group

Group 4: 1000 mg/kg group

Table 2: Body weights (g): Group Mean Values (Males)

Group/sex	Day						Change
	-7	0	7	14	21	28	0-28
1M	272	341	393	433	466	495	155
2M	262	328	379	416	445	472	144
3M	265	328	378	414	443	473	145
4M	269	335	387	421	448	477	142

Table 3: Body weights (g): Group Mean Values (Females)

Group/sex	Day (prior to mating)				Change	Day (during gestation)					Change	Day (during lactation)
	-7	0	7	14	0 - 14	0	7	14	16	20	0 - 20	1	4
1F	237	260	279	291	31	299	335	372	394	444	145	312	335
2F	232	255	268	282	28	285	324	360	382	433	148	304	331
3F	235	267	285	299	32	304	343	381	404	448	144	318	343
4F	231	255	270	285	30	288	326	362	387	438	150	312	338

 

Table 4: Clinical Chemistry (Group Mean Values)

Group / sex	ALP (U/L)	ALT (U/L)	AST (U/L)	LDH (U/L)	CPK (U/L)	Urea (mmol/L)	Glu (mmol/L)	T.Bil (µmol/L)	Chol (mmol/L)	TP (g/L)	Alb (g/L)	Glob (g/L)	AG-R	Na (mmol/L)	K (mmol/L)	Cl (mmol/L)	Phos (mmol/L)	Ca (mmol/L)	Crea (µmol/L)
1M	207	62	93	227	204	7.4	7.45	1.7	1.7	6.4	42	22	1.9	142	5.3	103	2.34	2.73	34
2M	183	61	91	192	168	6.5	8.07*	1.7	1.8	67	42	25	1.7	143	5.1	104	2.45	2.75	3.2*
3M	195	61	84	159	164	6.5	7.47	1.7	1.8	65	42	23	1.9	144	5.1	103	2.51	2.73	30#
4M	185	61	82	164	158	7.0	8.31#	1.7	1.8	67	42	25	1.7	143	5.0	104	2.42	2.75	31*
1F	81	91	75	198	122	8.8	5.71	1.8	2.1	62	41	22	1.9	142	5.2	104	1.6	2.69	45
2F	100	101	87	185	117	10.1	5.48	1.9	2.2	66*	42	24	1.8	141	5.5	103	2.0	2.73	47
3F	89	76	79	157	106	9.7	6.13	1.7	1.6	63	42	21	2.0	141	5.6	104	1.28	2.72	46
4F	100	90	93	148	104	10.4	6.07	1.7	2.2	67#	44	22	2.0	142	5.2	103	1.85	2.71	45

* p < 0.05

# p < 0.01

Conclusions:

In males, treatment with the test material at 100 mg/kg/day and above was associated with a slight decrease in creatine phosphokinase and treatment at 300 mg/kg/day and above was associated with a slight decrease in lactate dehydrogenase. In females, treatment with test material at 300 mg/kg/day and above was associated with slight decreases in creatine phosphokinase and lactate dehydrogenase.
These were the only clinical chemistry parameters affected and there were no other signs of toxicity noted in any other parameters or end points that were evaluated in this study. The toxicological significance of the differences in creatine phosphokinase and lactate dehydrogenase could not be established and therefore were considered not to be adverse.
In conclusion, under the conditions of this study the No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day.

Executive summary:

The oral repeat dose toxicity of the test material was determined in a GLP study which was conducted in accordance with the standardised guideline OECD 422. The study also placed an emphasis on neurological effects as a specific endpoint to identify any neurotoxic potential of the test material. During the study groups of 10 male and 10 female rats were administered test material at dose levels of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day. The males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (ca 4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca 6 weeks of treatment).

At dose levels up to 1000 mg/kg/day, there were no effects of treatment on clinical signs, body weights, body weight changes, food consumption, ophthalmology, detailed functional tests and observations, clinical pathology parameters (haematology and coagulation), gross necropsy findings, organ weights and histopathological examinations.

 

In males, treatment with the test material at 100 mg/kg/day and above was associated with a slight decrease in creatine phosphokinase and treatment at 300 mg/kg/day and above was associated with a slight decrease in lactate dehydrogenase. In females, treatment with the test material at 300 mg/kg/day and above was associated with slight decreases in creatine phosphokinase and lactate dehydrogenase.

 

These were the only clinical chemistry parameters affected and there were no other signs of toxicity noted in any other parameters or end points that were evaluated in this study. The toxicological significance of the differences in creatine phosphokinase and lactate dehydrogenase could not be established and therefore were considered not to be adverse.

 

In conclusion, under the conditions of this study the No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is a GLP study conducted on the registration substance and performed accordance with the standardised guideline OECD 422. The key study was assigned a reliability score of 1 according to the criteria of Klimisch (1997).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the key study, the oral repeat dose toxicity of the test material was investigated according to methods outlines in the standardised guideline OECD 422. The study placed an emphasis on neurological effects as a specific endpoint to identify any neurotoxic potential of the test material. During the study groups of 10 male and 10 female rats were administered test material at dose levels of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day. The males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (ca. 4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca. 6 weeks of treatment).

At dose levels up to 1000 mg/kg/day, there were no effects of treatment on clinical signs, body weights, body weight changes, food consumption, ophthalmology, detailed functional tests and observations, clinical pathology parameters (haematology and coagulation), gross necropsy findings, organ weights and histopathological examinations.

 

In males, treatment with the test material at 100 mg/kg/day and above was associated with a slight decrease in creatine phosphokinase and treatment at 300 mg/kg/day and above was associated with a slight decrease in lactate dehydrogenase. In females, treatment with the test material at 300 mg/kg/day and above was associated with slight decreases in creatine phosphokinase and lactate dehydrogenase.

 

These were the only clinical chemistry parameters affected and there were no other signs of toxicity noted in any other parameters or end points that were evaluated in this study. The toxicological significance of the differences in creatine phosphokinase and lactate dehydrogenase could not be established and therefore were considered not to be adverse.

 

In conclusion, under the conditions of this study the No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day.

 

The oral route is considered the most appropriate given the nature of the test substance it is considered unnecessary to undertake further repeated dose toxicity testing via the dermal or inhalation routes. Furthermore, the existing oral data is considered to adequately address the repeated dose toxicity endpoint and a further 90-day study is regarded as unnecessary.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
This is the only study available addressing this endpoint.

Justification for classification or non-classification

In accordance with criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for specific organ toxicity, repeated dose. The effects observed in the available study are not considered to be toxicologically significant and do not indicate any signs of organ dysfunction.