Neodymium di(2-ethylhexyl) phosphate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 24 May 2007 to 28 March 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: the study was performed according to internationally recognised guidelines and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

the homogeneity of the dosing solutions was not checked.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

see higher

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.43 (Neurotoxicity Study in Rodents)

Deviations:

yes

Remarks:

see higher

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050

Deviations:

yes

Remarks:

see higher

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: on the first day of treatment, the animals were approximately 6 weeks old
- Weight at study initiation: the males had a mean body weight of 208 g (range: 199 g to 220 g) and the females had a mean body weight of 167 g (range: 160 g to 179 g)
- Fasting period before study: yes
- Housing: the animals were housed individually in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm)
- Diet: free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 6557303 (SSNIFF Spezialdiäten GmbH, Soest, Germany), distributed weekly
- Water: free access to bottles containing tap water (filtered with a 0.22 μm filter)
- Acclimation period: for a period of 6 days before the beginning of the treatment period

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20%
- Air changes: approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12hrs dark / 12hrs light (7:00 - 19:00)

IN-LIFE DATES: from 30 May 2007 (first day of acclimation period) to 4 July 2007 (day of necropsy of last animal)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as suspension in the vehicle (corn oil). The test item was mixed with the required quantity of vehicle in order to achieve the concentration of 30, 90 and 200 mg/mL. The test item dosage forms were prepared daily and were stored at +4°C, protected from light, prior to use.
The dosage forms were stirred continuously throughout the dosing procedure. The dosage forms were allowed to come to room temperature for at least 15 minutes prior to administration to the animals and were administered at room temperature.

VEHICLE
- Justification for use and choice of vehicle: the test item is not soluble in water
- Concentration in vehicle: 30, 90 and 200 mg/mL
- Amount of vehicle: 5 mL/kg/day
- Lot/batch no.: 015K0115 and 065K0077, supplied by Sigma (Saint-Quentin-Fallavier, France)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test item was determined in samples of each control and test item dosage form prepared for use in weeks 1 and 4. Duplicate samples (2 x 5 mL) were taken and stored frozen (-20°C) until dispatch for analysis by ICP-OES.
All dosage form samples taken were kept frozen (at -20°C) until dispatch on completion of the study. Samples were sent to the lab on solid carbon dioxide.

A satisfactory agreement was observed between the nominal and actual concentrations of the test item in the administered dosage forms analyzed since the deviations from nominal concentrations remained within an acceptable range of +1% to +8%.

Duration of treatment / exposure:

29 days

Frequency of treatment:

once a day, at approximately the same time

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 150, 450 an 1000 mg/kg bw/day
Basis:
other: nominal in vehicle

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose-levels were selected on the basis of the results of a 7-day range-finding toxicity study by oral route performed in the same species (CIT/Study No. 32952 TSR), in which the same dose-levels were tested and no in-life effects were observed.
- Rationale for animal assignment: during the acclimation period, the required number of animals (20 males and 20 females) was selected according to body weight and clinical condition. They were then allocated to groups (by sex), using a computerized stratification procedure, so that the average body weight of each group was similar.

Positive control:

not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (morbidity and mortality) or once daily (clinical signs).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study.
- Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: at least once before group allocation, on the first day of treatment, and then once a week until the end of the study

FOOD CONSUMPTION:
- The quantity of food consumed by the animals in each cage was recorded once a week until the end of the study

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period (blood samples were taken from the orbital sinus of the animals, before the daily treatment)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (deprived of food for an overnight period of at least 14 hours)
- How many animals: all animals
- Parameters determined: Erythrocytes (RBC), Hemoglobin (HB), Mean cell volume (MCV), Packed cell volume (PCV), Mean cell hemoglobin concentration (MCHC), Mean cell hemoglobin (MCH), Thrombocytes (PLT), Leucocytes (WBC), Differential white cell count with cell morphology, neutrophils (N), eosinophils (E), basophils (B), lymphocytes and large unstained cells (L+LUC), monocytes (M), Prothrombin time (PT),

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period (blood samples were taken from the orbital sinus of the animals, before the daily treatment)
- Animals fasted: Yes (deprived of food for an overnight period of at least 14 hours)
- How many animals: all animals
- Parameters determined: Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (I.PHOS), Glucose (GLUC), Urea (UREA), Creatinine (CREAT), Total bilirubin (TOT.BIL.), Total proteins (PROT), Albumin (ALB), Albumin/globulin ratio (A/G), Total cholesterol (CHOL), Triglycerides (TRIG), Alkaline phosphatase (ALP), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at the end of the treatment period
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other: see detail below
The animals were randomized in order to ensure "blind" evaluation.
All animals were observed in the cage, in the hand and in the standard arena.
The following parameters were assessed and graded:
. "touch escape" or ease of removal from the cage,
. in the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
. in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.
Then, the following parameter measurements, reflexes and responses were recorded:
. touch response,
. forelimb grip strength,
. pupillary reflex,
. visual stimulus response,
. auditory startle reflex,
. tail pinch response,
. righting reflex,
. landing foot splay,
. at the end of observation: rectal temperature.
Finally, motor activity of all animals was measured once by automated infra-red sensor equipment over a 60-minute period.

OTHER:
- ESTROUS CYCLE monitoring: the stage of the estrous cycle of each female was determined from a fresh vaginal lavage (stained with methylene blue), each morning from day 22 until the day of sacrifice.
- SPERM ANALYSIS - Parameters examined in all male rats: testis weight (left), epididymis weight (left), epididymal sperm motility, sperm morphology and sperm count in epididymides and sperm count in testes

Sacrifice and pathology:

SACRIFICE:
On completion of the treatment period, after at least 14 hours fasting for the males, all animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination. Males were sacrificed on day 29, females were sacrificed on the day of diestrous (first sacrifice on day 28) or on day 32 if diestrous was not observed between days 28 and 32.

GROSS PATHOLOGY: Yes
The organs specified in the table 1 were weighed wet as soon as possible after dissection.
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

HISTOPATHOLOGY: Yes
A microscopic examination was performed on:
- all tissues listed in Table 1 for animals of the control and high-dose groups (groups 1 and 4) sacrificed at the end of the treatment period,
- all macroscopic lesions of all the animals of the low- and intermediate-dose groups (groups 2 and 3) sacrificed on completion of the treatment period.
- A detailed and careful microscopic examination was made of the right testis of each male and of the right ovary of each female in all groups, with knowledge of the stage of the spermatogenic and of the estrous cycles, respectively.

Statistics:

Statistical analyses were performed on body weight, food consumption, hematology, blood biochemistry and organ weight data.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY:
There were no unscheduled deaths.
4/5 males and 4/5 females treated at 1000 mg/kg/day had ptyalism. This was observed from week 2 or week 3 in the males and from week 3 or week 4 in the females and lasted until sacrifice. One male treated at 450 mg/kg/day and one male treated at 1000 mg/kg/day had chromodacryorrhea from day 14 to day 23 or on day 20. These effects were considered as non-adverse.
One female treated at 1000 mg/kg/day had loud breathing on days 22 and 23 but this was also observed in one control male on day 22 so was considered to be unrelated to treatment with the test item.

BODY WEIGHT AND WEIGHT GAIN (see table 2):
All male groups had mean body weight gains and body weights that were similar to the controls.
All female groups gained more body weight than the controls during the study but this did not significantly affect the mean body weights on day 28.

FOOD CONSUMPTION:
All male groups had similar mean food consumption to the controls.
Females treated at 1000 mg/kg/day had higher mean food consumption throughout the treatment period, when compared with the controls, achieving statistical significance over the intervals days 15 to 22 and days 22 to 28. The female group treated at 450 mg/kg/day also had slightly higher mean food consumption than controls from week 2. A relationship to treatment cannot be excluded although the effect was considered not to be adverse.

HAEMATOLOGY:
The mean white blood cell count of both males and females treated at 1000 mg/kg/day was lower than concurrent control values. This decrease was observed for all the types of white blood cell but achieved statistical significance for male basophil levels (p<0.05). This was considered to be related to treatment with the test item.
There were no effects on the red blood cell parameters (hemoglobin, hematocrit, red blood cell count, platelets) or coagulation (prothrombin time) in males or females treated at any dose and the effects on white blood cell count were not observed in males or females treated at 150 or 450 mg/kg/day.

CLINICAL CHEMISTRY:
Inorganic phosphorus levels were statistically significantly lower in females treated at 150 or 1000 mg/kg/day (-8% and -10%, p<0.05, respectively), however as the same values were obtained at 150 and 1000 mg/kg/day with no variation at 450 mg/kg/day this was considered to be spontaneous in origin and not treatment-related.
Cholesterol level was statistically significantly lower in males treated at 1000 mg/kg/day (-22%, p<0.05), however a decrease in cholesterol is not toxicologically relevant.
Electrolytes, glucose, urea, creatinine, bilirubin, proteins, triglycerides and liver enzymes were unaffected by treatment with the test item in males or females at any dose-level.

NEUROBEHAVIOUR:
- Motor activity:
The females treated at 450 mg/kg/day had slightly lower mean number of movements than the controls however no effects were observed at 150 or 1000 mg/kg/day therefore it was considered that treatment with the test item did not effect motor activity.

- Functional Observation Battery:
All animals achieved normal scores for touch escape, reactivity to handling, touch response, fur appearance, pupil size, grooming, palpebral closure, gait, posture, breathing, defecation, urination, visual stimulus response, pupillary reflex, auditory startle reflex, tail pinch response, righting reflex, forelimb grip strength and rectal temperature.
All animals had absence of salivation, lacrimation, piloerection, exophthalmos, tremors, twitches, clonic or tonic convulsions, hypoactivity or hyperactivity, ataxia, hypotonia, stereotypy and abnormal behavior.
5/5 males treated at 1000 mg/kg/day had a smaller landing foot splay than the lowest control value (group mean -37% compared to controls). This was considered to be related to treatment.
Females treated at 150 or 450 mg/kg/day also had a smaller landing foot splay (-41% and -22%, respectively). 3/5 females treated at 150 mg/kg/day and 2/5 females treated at 450 mg/kg/day had lower values than the lowest control value, although no effects were observed in the males treated at the same dose-levels and only 1/5 females treated at 1000 mg/kg/day showed a decrease. As this observation was not dose-related and it was noted only in females at 150 and
450 mg/kg/day and only 1/5 females treated at 1000 mg/kg/day were affected, it was considered not to be related to treatment with the test item in these animals.

ORGAN WEIGHTS (see table 3):
When compared to controls, there was a statistically significant increase in absolute and relative liver weights of the males given 1000 mg/kg/day. These changes were considered to be treatment-related.
A decrease in absolute heart weight was present in males treated at 450 or 1000 mg/kg/day. A relationship to the treatment with the test item was considered to be uncertain because of the poor dose-relationship, the absence of microscopic correlation, and because of the small magnitude of the heart weights changes.
There were no treatment-related organ weight changes in females given the test item at 150, 450 or 1000 mg/kg/day. The increase in ovaries weights observed in females treated at 450 mg/kg/day was of low magnitude, without any dose-relationship and thus was not considered to be related to the test item.
The other differences noted at the end of the treatment period in both sexes were slight, mainly non-statistically significant, without dose-relationship and were consequently not considered to be toxicologically relevant.

GROSS PATHOLOGY:
Macroscopic findings attributable to the treatment with the test item were observed in the forestomach of one female at 450 mg/kg/day (raised grey/white foci) and correlated with microscopic examination.
The few other gross findings noted in the males and females of all groups were those commonly recorded in the Sprague-Dawley rat and none were considered to be toxicologically relevant.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related findings were recorded in the forestomach from both sexes at 150, 450 or 1000 mg/kg/day (see table 4) and in the liver of the males at 450 or 1000 mg/kg/day.
In the forestomach from both sexes, multifocal epithelial cells hyperplasia that consisted of epithelial thickening with rete pegs and folds formation, increased number of epithelial cells layers was observed in 1/10, 5/10, 7/10 and 8/10 rats given the test item at 0, 150, 450 or 1000 mg/kg/day respectively. Erosions were noted in 2/10 rats from both sexes dosed at 450 or 1000 mg/kg/day. Hyperkeratosis was also present in 4/5 and 5/5 males treated at 450 and 1000 mg/kg/day respectively. Squamous crusts were observed in 4/5 male and 3/5 female rats treated at 450 mg/kg/day and in 3/5 males and 2/5 females treated at 1000 mg/kg/day. Submucosal inflammation that consisted of mixed inflammatory cells was found in 4/5 male and 2/5 female rats given the test item at 450 mg/kg/day and in 2/5 males and 2/5 females given the test item at 1000 mg/kg/day. These findings, most notably epithelium hyperplasia and crusts correlated with the necropsy finding (raised greyish/whitish foci) observed in the female treated at 450 mg/kg/day. These microscopic findings were considered to be treatment related and were suggestive of a local irritant effect of the test item.
In the liver, minimal focal subcapsular hepatocellular coagulative necrosis was observed in 1/5 and 3/5 males treated at 450 and 1000 mg/kg/day respectively. This change was considered secondary to slight increase in liver weights (with no microscopic correlate) and consequential compression of the hepatocytes adjacent to the capsule. This finding was considered of low toxicological importance.
All the other microscopic findings noted in the males and females that were examined at the end of the treatment period were those which are commonly recorded in the Sprague-Dawley rat kept under laboratory conditions and consequently were not considered to be toxicologically relevant.

OTHER FINDINGS:

ESTROUS CYCLE MONITORING:
All females in all groups were cycling normally.

SPERM ANALYSIS:
The percentages of motile sperm and morphologically normal sperm as well as the mean number of sperm heads in the testis and the mean daily sperm production rates were comparable with the controls for all treated groups. The mean number of sperm in the epididymis and the mean number of sperm per gram of epididymis was slightly lower at 150 and 1000 mg/kg/day than the controls.
The individual values were within the range of the individual control values (except one animal at 150 mg/kg/day for number of spermatozoa per cauda of epididymis) therefore this slight difference was considered to be unrelated to treatment with the test item.

EXAMINATION OF OVARIES AND TESTES:
Qualitative testis staging did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycles in males treated with the test item.
No alterations in estrous cycles were observed in females given the test item.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic toxicity (base on haematology, organ weight,; Functional Observation Battery)

2

Dose descriptor:

NOAEL

Effect level:

< 150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: local effect (based on microscopic findings in the forestomach)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 2 : Mean body weights and mean body weight changes (in g)

Sex	Male				Female
Dose level (mg/kg /day)	0	150	450	1000	0	150	450	1000
Day 1-28	+201	+202 (0%)	+200 (0%)	+187 (-7%)	+71	+82 (+15%)	+80 (+13%)	+77 (+8%)
Mean body weight on day 28	411	408 (-1%)	409 (0%)	395 (-4%)	236	247 (+5%)	250 (+6%)	246 (+4%)

 

Table 3 : Liver and heart weights (group mean values in grams)

Dose-level (mg/kg/day)	Group	Male
		Liver		heart
		Absolute	Body-relative	Absolute	Body-relative
0 150 450 1000	1 2 3 4	11.01 11.27 12.67 14.15 *	2.88 3.00 3.39 3.88 **	1.34 1.23 1.15 ** 1.16 **	0.353 0.327 0.307 * 0.319
		Female
0 150 450 1000	1 2 3 4	9.45 9.46 10.61 9.96	3.87 3.75 4.15 3.97	0.912 0.917 0.873 0.819	0.375 0.364 0.340 0.327

Statistically significant from controls: *: p<0.05, **: p<0.01.

 

Table 4:Summary incidence of grading in the forestomach of rats given the test item

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/day)	0	150	450	1000	0	150	450	1000
- Epithelial cell hyperplasia Grade 1 Grade 2 Grade 3	1 - -	3 - -	- 1 3	2 2 1	- - -	2 - -	- 2 1	1 - 2
Total affected Mean severity	1/5 1.0	3/5 1.0	4/5 2.8	5/5 1.8	0/5 -	2/5 1.0	3/5 2.3	3/5 2.3
- Erosion Grade 1 Grade 2	1 -	1 -	2 2	5 -	- -	- -	- 1	2 -
Total affected Mean severity	1/5 1.0	1/5 1.0	4/5 1.5	5/5 1.0	0/5 -	0/5 -	1/5 2.0	2/5 1.0
- Squamous crust(s) Grade 1 Grade 2	- -	- -	4 -	3 -	- -	- -	3 -	1 1
Total affected Mean severity	0/5 -	0/5 -	4/5 1.0	3/5 1.0	0/5 -	0/5 -	3/5 1.0	2/5 1.5
- Submucosal inflammation Grade 1 Grade 2	- -	- -	3 1	1 1	- -	- -	2 -	2 -
Total affected Mean severity	0/5 -	0/5 -	4/5 1.3	2/5 1.5	0/5 -	0/5 -	2/5 1.0	2/5 1.0

-: no animals affected.

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level based on local effects (NOAEL local) was less than 150 mg/kg/day for both male and female rats based on microscopic findings in the forestomach.
The No Observed Adverse Effect Level based on systemic effects (NOAEL systemic) is at 450 mg/kg/day for both male and female rats.

Executive summary:

In a subacute toxicity study scored as validity 1 according to Klimisch criteria (OECD guideline 407, GLP, CIT report No.32953 TSR, 2007) neodymium tris-(di-2-éthylhexyl phosphate) was administered daily to 5 Sprague-Dawley rats/sex/dose by gavage at dose levels of 0, 150, 450 or 1000 mg/kg/day as a suspension in corn oil.

During the study, animals were checked regularly for mortality and clinical signs, and body weights and food consumption were recorded.

Hematological and blood biochemical investigations as well as a Functional Observation Battery, including motor activity, were performed on all animals at the end of the treatment period.

Estrous cycles of the females were monitored for approximately 1 week prior to sacrifice.

At the end of the 4-week exposure period, the animals were killed and were submitted for a macroscopic and microscopic post-mortem examination. In addition, sperm analysis (epididymal and testicular sperm counts, sperm motility and morphology) was performed for each male and a detailed and careful microscopic examination of one testis and one ovary was performed for each animal.

 

No treatment-related of toxicological relevance was observed concerning the following effects: mortality (there were no unscheduled deaths), clinical signs, body weight and body weight gain, food consumption and blood chemistry. There were no treatment-related effects on sperm motility or morphology or on epididymal or testicular sperm count. All females had normal estrous cycles. There was no macroscopic finding except in the forestomach of one female treated at 450 mg/kg bw/day.

The males treated at 1000 mg/kg bw/day had a smaller landing foot spay than control but there were no other effects observed in the Functional Observation Battery (FOB) and motor activity and no effects were observed in the males from others treated groups and in females from all treated groups., The mean white blood cell count of both males and females treated at 1000 mg/kg bw/day was lower than concurrent control values. This decrease was observed for all the types of white blood cell but achieved statistical significance for male basophile levels (p<0.05). No effects on haematology parameters were observed in males and females from the others treated groups. There was a statistically significant increase in absolute and relative liver weights in male treated at 1000 mg/kg bw/day but no relevant differences to control values were observed in males from the other treated groups and females from all groups,

Multifocal epithelial cells hyperplasia of the forestomach with or without erosion, hyperkeratosis, squamous crust and submucosal inflammation were observed at a dose-related increased incidence and at a dose-related increased severity at all dose-levels in the animals of both sexes. This finding is considered to be related to treatment, although it is probably a local effect related to the administration of the test item by gavage.

 

Under the experimental conditions, it was therefore considered that:

- the No Observed Adverse Effect Level for local effects (NOAEL local) was less than 150 mg/kg/day based on microscopic findings in the forestomach.

- the No Observed Adverse Effect Level for systemic effects (NOAEL systemic) was 450 mg/kg/day based on the systemic effects observed (haematology, organ weight and one FOB parameter).

 

Based on this study, no classification for repeat-dose toxicity is warranted, according to the criteria of Annex VI Directive 67/548/EEC or UN/EU GHS.

 

 This study is classified as acceptable. It satisfies the OECD/EU guideline requirements on repeated dose toxicity testing.