Poly[oxy[(hydroxymethyl)-1,2-ethanediyl]], .alpha.-dodecyl-.omega.-hydroxy-

Endpoint summary

Description of key information

In a 4-week oral toxicity study performed similarly to OECD guideline 407 and in compliance with GLP, a NOAEL was identified at 150 mg/kg bw/day.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 June 2002 to 12 July 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study following OECD guideline 407. Well conducted and documented study with minor deviations: prostate of male rats was not weighed at the end of the treatment.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

prostate of male rats was not weighed at the end of the treatment.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

prostate of male rats was not weighed at the end of the treatment.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, l'Arbresle, France
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation: mean body weight for males: 228 g (range: 217-238 g) and for females: 174 g (range: 159-193 g)
- Housing: individually in suspended wire mesh cages
- Diet (e.g. ad libitum): A04 C pelleted maintenance diet (UAR, Villemoisson, Epinay-sur-Orge, France), ad libitum
- Water (e.g. ad libitum): filtered tap water, ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 12 cycles, non recycled air
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

IN-LIFE DATES: From: 4 June 2002 To: 12 July 2002

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified by reverse osmosis

Details on oral exposure:

Method of administration: Oral gavage

VEHICLE
- Concentration in vehicle: 10, 30 or 120 mg/mL
- Amount of vehicle (if gavage): constant dosage volume of 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the preparations were checked. Concentrations were measured by gas liquid chromatography with flame ionization detection. Actual concentrations of the administered dosage were within ± 8% of the theoretical value.

Duration of treatment / exposure:

29 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
150 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
600 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: on the basis of the results of a 7-day range finding toxicity study performed in the same species at 150, 450 and 1000 mg/kg bw/day

Positive control:

Not applicable

Observations and examinations performed and frequency:

MORBIDITY and MORTALITY:
At least twice a day during the treatment period.

CLINICAL SIGNS:
At least once a day, at approximately the same time.

DETAILED CLINICAL OBSERVATIONS:
Detailed clinical observations were performed in all animals outside the home cage, in a standard arena, 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-mutation, walking backwards) were also recorded.

BODY WEIGHT:
The body weight of each animal was recorded at least once before allocation of the animal to groups, on the first day of the treatment and then once a week until the end of the study.

FOOD CONSUMPTION:
Recorded once a week for each animal, over a 7-day period.

HAEMATOLOGY:
Blood samples were taken from the orbital sinus of the animals (before the daily treatment). Prior to blood collection, the animals were deprived of food for an overnight period of at least 14 h. Paramaters examined were: erythrocytes, hemoglobin, mean cell volume, packed cell volume, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocytes, leucocytes, differential white cell count with cell morphology, neutrophils, eosinophils, basophils, lymphocytes, monocytes and prothrombin time.

CLINICAL CHEMISTRY:
Blood samples were taken from the orbital sinus of the animals (before the daily treatment). Prior to blood collection, the animals were deprived of food for an overnight period of at least 14 h. Parameters measured were sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin ratio, cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase

NEUROBEHAVIOURAL EXAMINATION:
All animals were evaluated once at the end of the treatment period.
This included a detailed clinical examination, measurement of reactivity to manipulation or to different stimuli and motor activity.
- Battery of functions tested: touch escape or ease of the removal from the cage; in the hand: fur appearence, salivation, lacrymation, piloerection, exophthalmia, reactivity to handling, pupil size (presence of myosis or mydriasis); in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, convulsions, gait, arousal (hypo- and hyperactivity), posture, stereotypes, behaviour, breathing, ataxia and hypotonia. Then, the following parameter measurements, reflexes and responses were recorded: touch response, forelimb grip strength, pupil reflex, visual stimulus, 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 by automated infra-red sensor equipment (not validated) over a 15-minute period.

Sacrifice and pathology:

GROSS PATHOLOGY: A complete macroscopic post-mortem examination was performed on all study animals. Several organs were weighed (see table 1).

HISTOPATHOLOGY: Yes: all macroscopic lesions of the low and intermediate dose, following tissues of the high dose and the control groups (see table 1). For all animals, tissues were preserved in 10% buffered formalin (except for the eyes and Harderian glands which were fixed in Davidson's fixative, and the testes and epididymides which were preserved in Bouin's fluid). All tissues required for microscopic examination were embedded in paraffin wax, sectioned at a thickness of approximately 4 microns and stained with hematoxylin-eosin.

Other examinations:

No data

Statistics:

Statistical analysis for body weight, haematology, blood biochemistry, food consumption and organ weight was performed.

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:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Thickened mucosa in the forestomach in one female given 150 mg/kg bw/day and in one male and two females given 600 mg/kg bw/day; mucosa with several depressed area in 1/5 male given 600 mg/kg bw/day.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological changes in the forestomach (epithelial cell hyperplasia, hyperkeratosis, submucosal edema and erosion or ulceration) in a dose-related manner

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Loud or abdominal breathing was noted in 3/5 males and 2/5 females given 600 mg/kg/day. In view of the apparent irritant properties of the test item, this sign was attributed to the treatment with the test item.
Ptyalism was observed in 5/5 males and 3/5 females given 150 mg/kg/day and in 5/5 males and 5/5 females given 600 mg/kg/day. This sign which is commonly noted with test items administered by gavage was not considered as an adverse effect.
No mortalities occured during the treatment period.

BODY WEIGHT AND WEIGHT GAIN
The differences observed especially in the males were slight, not dose-related and nor correlated with the food consumption.

FOOD CONSUMPTION
No differences of toxicological significance were observed in the food consumption of the treated animals.

HAEMATOLOGY (Table 2)
The differences observed (i.e. white blood cell count, reticulocyte count, hemoglobin level, packed cell volume and prothrombin time) were slight, not dose-related and with the individual values within the range of our historical background data.

CLINICAL CHEMISTRY
Compared to controls, a slightly higher mean inorganic phosphorus level was noted in males given 150 and 600 mg/kg bw/day (3.44 and 3.11 mmol/L vs. 2.89 mmol/L, respectively). The individual values in all males given 150 mg/kg bw/day and in 3/5 males given 600 mg/kg bw/day were above the upper limit of our historical background data (1.71 - 2.99 mmol/L). This slight increase was most probably factitious and of no toxicological importance, since these differences were not dose-related, lacked similar trend in the females, the individual value of one control animal was also above the upper limit of historical background data, and there were no any abnormalities in the related parameters (such as renal injury or hypercalcemia).

A slightly, statistically significant, higher mean alanine aminotransferase activity was observed in males given 600 mg/kg bw/day (32 IU/L vs. 16 IU/L). Since 4/5 of the individual values of the treated males were in the range of historical background data (9-33 IU/L) and as no histological changes were found in the liver, the higher mean alanine aminotransferase activity was considered to be of no toxicological relevance.

A slightly lower mean glucose level was observed in females given 50 and 150 mg/kg bw/day (4.07 and 4.19 mmol/L vs. 5.38 mmol/L).
The other differences observed (i.e. chloride and urea levels) were slight and/or not dose-related and with the individual values within the range of our historical background data. Although the differences in glucose levels were statistically significant and the individual values outside the range of historical background data (4.82 - 9.00 mmol/L), and since no similar findings were noted at the higher dose-level (600 mg/kg bw/day), they were considered to be without relationship to the treatment with the test item.

NEUROBEHAVIOUR
There was no evidence of disturbance of either autonomic or physiological functions at any dose-level. The functional test battery showed no treatment-related changes in any neurotoxicological parameter.
No relevant differences in motor activity were noted between control and treated animals.

ORGAN WEIGHTS
The most important differences in the mean weights were for the liver and the thyroids. As these weight differences were the contribution of few individuals and were without relevant histopathological abnormalities, they were considered to be of no toxicological importance.
Other differences were noted in organ weights between treated and control animals. However, these differences were generally slight, neither dose-related, nor showing the same trend in the two sexes and/or with variations in the individual values within the groups and between groups. They were thus also considered to be of no toxicological importance.

GROSS PATHOLOGY
The necropsy findings which were considered to be treatment-related occurred in the forestomach, as follows:
- thickened mucosa in 1/5 females given 150 mg/kg/day and in 1/5 males and 2/5 females given 600 mg/kg/day,
- mucosa with several depressed areas in 1/5 males given 600 mg/kg/day.
All these findings correlated with the epithelial cell hyperplasia and/or submucosal edema.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related microscopic changes in the fore stomach are shown in table 3.
The few other microscopic changes noted were recognized as those commonly recorded spontaneously in the untreated laboratory rat of this strain and age and to be of no toxicological importance.

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: At 600 mg/kg bw/day ptyalism, loud and abdominal breathing, significant histopathological changes in the forestomach

Critical effects observed:

not specified

Table 2: significant changes in hematology parameters at day 28

		Dose (mg/kg bw/d)	0	50	150	600
Females
	RBC T/L	M (2) SD N	7.61 0.296 4	7.17 0.233 5	6.96 * 0.415 5	7.39 0.161 5
	HB g/dL	M (2) SD N	15.2 0.29 4	14.0 * 0.32 5	13.9 ** 0.47 5	14.3 0.30 5
	PCV L/L	M (2) SD N	0.43 0.010 4	0.41 0.011 5	0.39 ** 0.011 5	0.41 0.007 5
Males	PT s	M (1) SD N	18.3 2.41 5	15.4 2.44 5	14.9 * 1.91 5	14.9 * 0.79 5

Significance of the difference between treated and control groups * P<0.05; ** P<0.01

(1): Dunnett test

(2): Dunn test

Table 3: microscopic findings in the forestomach

Dose (mg/kg bw/d)	0		50		150		600
Number of animals with microscopic findings in the forestomach	M	F	M	F	M	F	M	F
Inflammatory cells mucosa	5/5	5/5	5/5	5/5	5/5	5/5	5/5	5/5
Epithelial cell hyperplasia	-	-	1/5	1/5	1/5	1/5	3/5	3/5
Hyperkeratosis	-	-	-	-	1/5	1/5	2/5	2/5
Submucosal inflammatory cells	-	2/5	2/5	2/5	5/5	3/5	2/5	4/5
Submucosal edema	-	-	1/5	-	1/5	2/5	3/5	4/5
Erosion	-	-	-	-	-	-	-	1/5
Ulceration	-	-	-	-	-	1/5	-	-

Conclusions:

The minimal incidence and severity of the forestomach lesions in the animals given 50 and 150 mg/kg bw/day were not considered as adverse effects but probably the consequence of the local irritation during the gavage. Therefore the NOAEL was set at 150 mg/kg bw/day.

Executive summary:

In a 4-week oral toxicity study performed similarly to OECD guideline 407 and in compliance with GLP, Chimexane NV, mixed in purified water at dose levels of 50, 150 and 600 mg/kg bw/day, was administered by oral gavage to 3 treated groups of 5 Sprague-Dawley rats/sex/dose for 29 days. An additional group of 5 animals/sex received vehicle only.

The animals were checked daily for mortality and clinical signs. Detailed clinical observations were performed before the beginning of the treatment period and then once a week thereafter. Body weight and food consumption were recorded once a week during the study. Neurotoxic evaluation using functional observation battery (FOB) was carried out at the end of the treatment period. Hematology and blood biochemical investigations were performed at the end of the treatment period. On completion of the treatment period, the animals were killed and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from animals of the control and high-dose groups and the forestomach of all animals.

No mortality occurred during the study. Loud or abdominal breathing was noted at 600 mg/kg bw/day and ptyalism in several animals at 150 mg/kg bw/day and in all animals at the highest dose. There were no treatment-related changes in autonomic, physiological or neurotoxicological parameters. There were also no relevant treatment-related differences between control and treated animals in body weight, food consumption, hematology, blood biochemistry and organ weights. At macroscopic level, thickened mucosa in the forestomach was noted in one female given 150 mg/kg/day and in one male and two females at the highest dose level. At microscopic level, treatment-related changes were observed in the forestomach: minimal epithelial cell hyperplasia and/or submucosal edema in one male and one female at 50 mg/kg bw/day, minimal to slight changes (epithelial cell hyperplasia, hyperkeratosis, submucosal edema and erosion or ulceration) in one male and one female at 150 mg/kg bw/day, and minimal to moderate epithelial cell hyperplasia in three males and three females given 600 mg/kg/day, associated with hyperkeratosis in two males and two females.

Taking into account the minimal incidence and severity of the forestomach lesion at 50 and 150 mg/kg bw/day, the NOAEL was set at 150 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP study following OECD guideline 407. Well conducted and documented study with minor deviations.

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 a 4-week oral toxicity study performed similarly to OECD guideline 407 and in compliance with GLP, loud or abdominal breathing was noted at the highest dose tested (600 mg/kg bw/day). In the forestomach, minimal to moderate epithelial cell hyperplasia in three males and three females associated with hyperkeratosis in two males and two females were observed at this dose. Only minimal epithelial cell hyperplasia and/or submucosal edema were recorded in one male and one female at 50 mg/kg bw/day, and minimal to slight changes (epithelial cell hyperplasia, hyperkeratosis, submucosal edema and erosion or ulceration) were also observed in one male and one female at 150 mg/kg bw/day. Taking into account the minimal incidence and severity of the forestomach lesion at 50 and 150 mg/kg bw/day, the NOAEL was set at 150 mg/kg bw/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only one study available for this endpoint

Justification for classification or non-classification

As the NOAEL identified in the repeated dose toxicity study is higher than 100 mg/kg bw/day, there is no need for classification of Chimexane NV for repeated dose toxicity according to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272 -2008).