Amines, C12-14-alkyldimethyl

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 23 MAR 2010 to 19 MAY 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline Study (OECD 422) and according to GLP. Justification for read-across see chemical safety report chapter 1.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

As not every positive mating sign results in pregnancy, the mating period was extended until a positive mating sign was noted for all females (up to 17 days). This additional mating period was conducted to guarantee at least 8 pregnant females per group.

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemical Law and OECD Principles of GLP

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Amines, C12-18-alkyldimethyl, N-oxides

EC Number:

273-281-2

EC Name:

Amines, C12-18-alkyldimethyl, N-oxides

Cas Number:

68955-55-5

IUPAC Name:

dimethyl(pentadecyl)amine oxide

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Research Models and Services Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 10 weeks
- Weight at study initiation: Males 327.9 - 424.0 g; females 212.7 - 280.0 g
- Housing: Except during the mating period, the animals were kept singly in MAKROLON cages (type III) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 15 cm.
- Diet: Ad libitum
- Water: tap water ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +- 3°C (maximum range)
- Humidity (%): Relative humidity of 55% - 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23-03-2010 to 19-05-2010

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was suspended in the vehicle (tap water) to the appropriate concentrations and was administered orally at a constant application volume of 10 mL/kg b.w./day. The test item-diet mixture was freshly prepared every day.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg b.w./day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At study initiation:
Analysis of stability and concentration: Immediately after preparation of the mixtures as well as 8 and 24 hours after storage of the test item preparations at room temperature (3 samples/dose level group). Total number of samples: 9.
Analysis of Homogeneity: At the start of administration, during (middle) administration and before administration to the last animal of each dose level group (3 samples/dose level group). Total number of samples: 9.

At study termination:
Analysis of concentration: During treatment with the test item always before administration to the last animal/dose level group (1 sample/dose level group). Total number of samples: 3.
Test item-vehicle mixtures (in total 21 vials), 4 further vials containing the test item or vehicle of test days 1 and 42 were dispatched on dry ice by courier for analysis.

Duration of treatment / exposure:

Main study males (mated):
50 % of the main study males were dosed for 31 days and the other 50 % of the main study males were dosed for 36 days, depending if they were sacrificed on test day 32 or 37. This dosing period includes the pre-mating period (2 weeks), the mating period (1 to 17 days) and the post mating period up to and including the day before sacrifice (terminal sacrifice was conducted on test day 32 or 37.

Main study females (mated):
The main study females were dosed for 41 to 56 days. This period includes 2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.

Satellite animals (not mated animals):
The satellite animals were treated for 41 days followed by a recovery period of 16 days. The animals were not mated. Treatment started on the same day as of the main study animals and was conducted up to the day before the first scheduled sacrifice of the main study dams on test day 42.

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 40, 100 or 200 mg/kg b.w./day
Basis:
actual ingested

No. of animals per sex per dose:

The animals were randomly allocated to the 4 test groups as follows:

Group test substance No. and sex
dose of animals
[mg/kg b.w./day]# MS+RP

1 0 10 m + 5 m
(control) 10 f + 5 f

2 40
(low dose) 10 m 10 f

3 100
(intermediate dose) 10 m 10 f

4 200 10 m + 5 m
(high dose) 10 f + 5 f

MS: main study; animals scheduled for the reproduction study
RP: recovery period; satellite animals scheduled for a 16-day recovery period, the
animals were not mated and thus not used for the reproduction study
m: males
f: females
#: A correction factor of 3.13 was used.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on available toxicological data and a preliminary dose-range finding study (LPT study no. 25122). No mortality was noted in this dose-range-finding study no. 25122. Signs of systemic intolerance were noted in the animals of both sexes starting at 40 mg Aromox B-W 500/kg b.w./day. A slightly to severely reduced mean food consumption and mean body weight were noted in the male rats starting at 100 mg/kg b.w./day or at 250 mg/kg b.w./day, respectively. The females were not affected.
Necropsy revealed a thickened cardiac region of the stomach in one male rat treated with 250 mg/kg b.w./day as well as in all high dosed male and female rats treated with 400 mg Aromox B-W 500/kg b.w./day.

- Rationale for animal assignment: Random. At commencement of the study, the weight variation of animals used was minimal and did not exceed 20% of the mean weight of each sex.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily throughout the test period.
- Cage side observations included: Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality. Any signs of illness or reaction to treatment were recorded for each individual animal. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded. Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals. Detailed clinical observations were made in all male main study animals until terminal sacrifice in test week 5 or 6, respectively, in all female main study animals until the day of parturition and in all male and female satellite animals until terminal sacrifice in test week 8. These observations were made outside the home cage in a standard arena at the same time, each time. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and 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 behaviour (e.g. self-mutilation, walking backwards) were also recorded.
Mortality: Further checks were made early in the morning and again in the afternoon of each working day to identify dead or moribund animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily throughout the test period.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of dosing, weekly thereafter and at study termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum during lactation. Body weights were recorded individually for each adult animal. Live pups were weighted individually within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum during lactation.

FOOD CONSUMPTION:
The quantity of food left by individual animals was recorded on a weekly basis through-out the experimental period with the execution of the mating period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Drinking water consumption was monitored daily by visual appraisal throughout the study.

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period and at the end of the recovery period.
- Anaesthetic used for blood collection: Yes, light ether anaesthesia
- Animals fasted: Yes, fasted overnight
- How many animals:
At the end of the pre-mating period: 5 male and 5 female main study animals randomly selected of each group
At the end of the recovery period: All satellite animals
- Parameters checked: Haemoglobin content (HGB), erythrocytes (RBC), leucocytes (WBC), reticulocytes, platelets, differential blood count (relative), differential blood count (absolute), haematocrit value, thromboplastin time, activated partial thromboplastin time, mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period and at the end of the recovery period.
- Animals fasted: Yes, fasted overnight
- How many animals:
At the end of the pre-mating period: 5 male and 5 female main study animals randomly selected of each group
At the end of the recovery period: All satellite animals
- Parameters checked: Albumin, globulin, albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea (blood urea), calcium, chloride, potassium, sodium, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase.

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: See clinical signs

OTHER:
Neurological screening:
Screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) as well as the assessment of grip strength and motor activity assessment were conducted in five males and five females randomly selected from each main study group. The tests were conducted towards the end of the main study, 1 - 2 hours after dosing and before any blood sampling for labo-ratory examinations.

Observational screening
Males: Shortly before scheduled sacrifice (test day 29)
Females: During lactation, shortly before scheduled kill (test days 42, 43 or 56)
Righting reflex:
The animal was grasped by its tail and flipped in the air (approx. 60 cm) above the cart surface so that it turned head over heels. The normal animal should land squarely on its feet. If it landed on its side, 1 point was scored; if it landed on its back, 2 points were scored. This test was repeated five times and the total scores were recorded.
Body temperature:
An electronic probe thermometer (with a blunt probe) was used to take a rectal temperature, being allowed to equilibrate for 30 seconds before the reading was recorded.
Salivation:
Discharge of clear fluid from mouth, most frequently seen as beads of moisture on lips in rats. Normal state was to see none, in which case the score sheet space was left blank. If present, a plus sign was recorded in blank.
Startle response:
With the animal on the cart, the metal cage was struck with the blunt probe. The normal animal should exhibit a marked but short-lasting response, in which case the space on the scoring sheet was left blank. If present, a plus sign was entered.
Respiration:
While at rest on the cart, the animal's respiration cycle was observed and evaluated in terms of a scale from 1 (reduced) to 5 (increased), with 3 being normal.
Mouth breathing:
Rats are normally obligatory nose-breathers. Each animal was observed, whether it was breathing through its mouth or not (if it was, a check was placed in the appropriate box).
Urination:
When an animal was removed from its cage, the pan beneath the animal's cage was examined while returning the animal to its cage. The signs of urination were evaluated on a scale of 0 (lacking) to 5 (polyuria).
Convulsions:
If clonic or tonic convulsions were observed to occur, they were graded on intensity (from 1 (minor) to 5 (marked)) and the type and intensity were recorded.
Pilo-erection:
The fur of the animal's back was observed, whether it was raised or elevated. In the normal case (no pilo-erection) the space blank is left. If pilo-erection was present, a plus sign was entered in the blank.
Diarrhoea:
In examining the pan beneath an animal's cage, it was noted if there were any signs of loose of liquid stools. Normal state was for there to be none (0), in case of diarrhoea the intensity was recorded on a scale of 1 (slight) to 5 (much increased).
Pupil size:
The pupils were determined if they were constricted or dilated and were graded on a scale of 1 (constricted) to 5 (dilated), respectively, 3 being normal.
Pupil response:
The beam of light from the pen light was played across the eyes of the animal and the changes in pupil size were noted. In the normal animal, the pupil was constricted when the beam was on it and then dilated back to normal when the light was removed. It was noted if there was no response (in which case a minus sign was recorded in the blank space).
Lacrimation:
The animal was observed for the secretion and discharge of tears. In rats the tears contain a reddish pigment. No discharge was normal and in this case the box was left blank. If the discharge was present, a plus sign was entered.
Impaired gait:
The occurrence of abnormal gait was evaluated. The most frequent impairments were waddling (W), hunched gait (H), or ataxia (A, the inability of all the muscles to act in unison). The extend of any impairment was recorded on a scale of 1 (slight) to 5 (marked).
Stereotypy:
Each animal was evaluated for stereotypic behaviour (isolated motor acts or partial sequences of more complex behavioural patterns, occurring out of context and with an abnormal high frequency). These were graded on a scale of 0 to 5 if such signs were present.
Toe pinch:
The blunt probe was used to bring pressure to bear on one of the digits of the hindlimb. This should evoke a response from the normal animal, graded on a scale from 1 (ab-sent) to 5 (exaggerated).
Tail pinch:
The procedure detailed above was utilized with the animal's tail instead of its hindlimb and was graded on the same scale.
Wire manoeuvre:
The animal was placed on the metal rod suspended parallel to the cart approx. 60 cm above it. Its ability to move along the rod was evaluated. If impaired, a score of from 1 (slightly impaired) to 5 (unable to stay on wire) was recorded.
Hind leg splay:
Using an ink pad, the hind paws were marked with ink. The rat was then held 30 cm above a sheet of blotting paper on the cart. The animal was dropped and the distance between the prints of the two hind paws was measured.
Positional passivity:
When placed in an awkward position (such as on the edge of the top of the wire bot-tomed cage) on the cart surface, it was examined whether the animal immediately moved into a more normal position. If not, a score was recorded on a scale of 1 (slightly impaired) to 5 (cataleptic).
Tremors:
Periods of continued fine movements, usually starting in the limbs (and perhaps limited to them). The normal case is to have none, in which case no score was recorded. If present, they were graded on a scale of 1 (slight and infrequent) to 5 (continuous and marked).
Positive geotropism:
The animal was placed on the inclined (at an angle of approx. 30°) top surface of the wire cage with its head facing downward. It should turn 180° and face 'uphill', in which case the space on the form should be left blank. If this did not occur, a negative sign was recorded.
Limb rotation:
One of the animal's hindlimbs was taken and moved through its normal plane of rota-tion. In the normal state, it should rotate readily but there should be some resistance. The variations from normal were from no resistance (1) to markedly increased resis-tance or rigidity (5), with 3 being normal.
Auditory function:
Each animal was placed into a container and observed for Preyer's reflex (twitching of the pinna) in response to a high frequency sound stimulus. The stimulus was repeated, if necessary, up to 3 times.

Functional tests
Grip strength:
Prior to testing, the gauge (Chatillon, Modell DPP - 1.0 kg) was calibrated with a set of known weights and the apparatus adjusted for the size of the animal (about 1 cm clearance on both sides of the animal). After the strain gauge was zeroed and set in the record mode, the animal was placed into the trough with the forepaws inside the triangular grasping ring. Using one hand, the animal was grasped about 2.5 cm of the way up toward the base of the tail and steadily pulled (approx. 2.5 cm/sec) away from the ring until the grip was broken. The animal continued to be pulled along the trough until the hindlimbs grasped the T-bar. The trial was completed when grip of the hindlimbs was broken. Three successive readings were taken for each animal with an inter-trial interval long enough to record the data and zero both meters for the next trial.

Locomotor activity
The rats were individually placed into a motility system with microprocessor control and automatic statistical evaluation. Each unit had 2 individually adjustable channels so that slight static movements and active moving could be differentiated and separately scored (TSE Systems, Bad Homburg).
The motility meter created a magnetic field, movement of the animals interfered with the magnetic field. Two separate magnetic fields allowed two sensitivity levels to be chosen to distinguish between two types of movements:
High sensitivity: stereotype, static movement
(slight movements) (e.g. grooming, a stationary movement of the animal without leaving its own position)

Low sensitivity: active locomotion
(active moving)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
50 % of the male main study animals were sacrificed in a randomised way after a total dosing period of 31 days on test day 32, the other half of the male main study animals were sacrificed after a total dosing period of 36 days on test day 37. Dams with offspring were sacrificed on day 4 post-partum, or shortly thereafter. Females which did not deliver were sacrificed on the fourth or sixth day after the calculated day of delivery.
Dissection of all animals allocated to the recovery period was performed on test day 58.
At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory re-productive organs were recorded.
The weights of the following organs of all adult male animals were determined before fixation: Epididymis (2), testicle (2)
The weights of the following organs of in total 20 adult males and 20 adult females (5 animals/sex/main study group; randomly selected of each main study group, including not pregnant animals) and all satellite animals were determined before fixation: Adrenal gland (2), kidney (2), spleen, Brain, liver, thymus, heart, ovary.
Adrenal glands, gonads and kidneys were weighed individually and identified as left or right.

HISTOPATHOLOGY: Yes
The following organs or parts of organs of the randomly selected 20 male and 20 female animals (5 animals/sex/main study group) and all satellite animals were preserved in 7% Formalin:
Adrenal gland (2)
Bone marrow (os femoris)
Brain (cerebrum, cerebellum, brain stem)
Gross lesions observed
Heart (right and left ventricle, septum)
Intestine, large (colon, rectum)
Intestine, small (duodenum, jejunum,
ileum, incl. Peyer's patches, Swiss roll
method)
Kidney and ureter (2)
Liver
Lungs (with mainstem bronchi and
bronchioles [preserved by inflation
with fixative and then immersion])
Lymph node (cervical) (1)
Lymph node (mesenteric) (1)
Nerve (sciatic)
Oesophagus
Seminal vesicle
Spinal cord (3 sections)
Spleen
Stomach
Thymus
thyroid (incl. parathyroids)
Tissue masses or tumours
(including regional lymph nodes)
Tongue (incl. base)
Trachea (incl. larynx)
Urinary bladder
In addition, the following organs or parts of organs of the reproductive system of all adult male and female animals were preserved in 7% Formalin; the testes and epididymides were preserved in Bouin's fixative:
Epididymides (2)
Mammary gland
Ovary (2)
Prostate
Testicle (2)
Uterus (incl. Cervix and oviducts)
Vagina
The afore-listed organs of the randomly selected parental animals of groups 1 and 4 (5 male and 5 female animals per group) and all satellite animals (in total 20 satellite animals) were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining.
The afore-listed organs of the reproductive system of all main study animals of groups 1 and 4 (in total 40 main study animals) and all satellite animals (in total 20 satellite animals) were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining.
Any other organs displaying macroscopic changes were also preserved.
Detailed histopathologic examination was performed on the ovaries, testes and epididymides (with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure) of the animals of the highest dose group (group 4) and the control group (group 1) following H.& E. and PAS staining.
In addition, frozen sections of the heart, liver and one kidney were made and stained with scarlet R. Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plan of section and in all cases where they were noted as grossly enlarged.
Due to test item-related changes, the following organs of 5 male and 5 female animals of the low and intermediate dose level groups (groups 2 and 3) were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining: Forestomach, lymph node (1, mesenteric)

Statistics:

The test item-treated groups 2 to 4 were compared with the control group 1.
STUDENT's t-test: All numerical functional tests (p ≤ 0.01)
The following limits were used:
p ≤ 0.01 ≙ t = 2.878 (18 degrees of freedom)
p ≤ 0.01 ≙ t = 2.921 (16 degrees of freedom)
p ≤ 0.01 ≙ t = 2.947 (15 degrees of freedom)
p ≤ 0.01 ≙ t = 3.355 (8 degrees of freedom)

Multiple t-test based on DUNNETT New tables for multiple comparisons with a control: Body weight / food consumption / haematology / clinical biochemistry /organ weights (absolute and relative) (p ≤ 0.01).
The following limits were used:
p ≤ 0.01 ≙ t = 3.09 (36, 33, 32 and 31 degrees of freedom)
p ≤ 0.01 ≙ t = 3.36 (8 degrees of freedom)
p ≤ 0.01 ≙ t = 3.39 (16 degrees of freedom)

For all numerical values (body weight, food consumption and organ weight data) gen-erated from the start of the mating period onwards homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT's t-test was carried out, limit of significance was p ≤ 0.01.

Exact test of R.A. FISHER: Histopathology (p ≤ 0.05)
For the comparison of classification measurements (for example the fertility index) the FISHER's exact test, n < 100 or chi 2-test with Yates' correction for continuity, n ≥ 100 (p ≤ 0.05) were employed.
These statistical procedures were used for all data. Significantly different data are indicated in the tables.

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 efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Main study / satellite animals: No test item-related mortality was noted. No signs of systemic toxicity were noted for the male and female animals treated with 40 mg test substance/kg b.w./day. Starting at 100 mg test substance/kg b.w./day increased salivation was noted for a very few male and female animals at 100 mg test substance/kg b.w./day and most animals at 200 mg test substance/kg b.w./day during all periods of the study (pre-mating, mating, post-mating, gestation and/or lactation period, respectively) starting on test day 1. Further, at 200 mg test substance/kg b.w./day laboured breathing was observed for one male and rough fur was noted sev-eral females during the pre-mating, mating, gesta-tion and/or lactation period, respectively.

Functional observations (carried out 1 to 2 hours after administration):
Main study animals: No influence was noted on the parameters of the functional observations at any of the tested dose levels for the males.
Females treated with 200 mg test substance/kg b.w./day revealed salivation and pilo-erection.
In addition, hindlimb grip strength of the females was reduced (statistically significant at p ≤ 0.01) by up to 71 % starting at 40 mg test substance/kg b.w./day, though no dose-response relationship was noted. A slight but not significant reduction was also observed for the males at 100 and 200 mg test substance/kg b.w./day.

BODY WEIGHT AND WEIGHT GAIN
Main study / satellite animals: No influence was noted on the body weight of main study and satellite animals during the entire study after treatment with 40, 100 or 200 mg test substance/kg b.w./day.

FOOD CONSUMPTION/WATER CONSUMPTION
Main study / satellite animals:No test item-related influence was observed on the food consumption of the male and female animals treated with either 40 or 100 mg test substance/kg b.w./day during the pre-mating, ges-tation and/or lactation period, respectively.
Treatment with 200 mg test substance/kg b.w./day resulted in a food intake statistically sig-nificant reduced by 11% in test week 2 (pre-mating period) for the male main study animals and for the female main study animals by 10% in test week 1 (pre-mating period) and by 13% on gestation day 7 (gestation period) compared to the control.
The food intake of the female satellite animals treated with 200 mg test substance/kg b.w./day was statistically significant reduced by 20% in test week 1 compared to the control. No influence was noted on the visual appraisal of the drinking water consumption at any of the tested dose levels.

HAEMATOLOGY
Main study animals: No test item-related changes in haematological parameters were noted at the end of the pre-mating period (test day 15) of male and female rats treated with 40 or 100 mg test substance/kg b.w./day compared to the control. Changes in haematological parameters were noted for the animals treated with 200 mg test substance/kg b.w./day: Increased neut (relative and absolute). This was statistically significant (p ≤ 0.01) for neut absolute in females.

CLINICAL CHEMISTRY
Main study animals:
No test item-related changes were noted on bio-chemical parameters on test day 15 of female rats treated with 40 mg test substance/kg b.w./day compared to the control.
For the male and/or female animals treated with 40, 100 or 200 mg test substance/kg b.w./day the following changes in biochemical parameters were observed: Increased ALAT statistically significant (p ≤ 0.01) for both males and females. However no histopathological changes were noted. Therefore the changes observed may be coinidental.

ORGAN WEIGHTS
Main study animals: No test item-related influence was noted.

GROSS PATHOLOGY
Main study animals: Macroscopic inspection at necropsy revealed no test item-related changes in the organs or tissues after treatment with either 40, 100 or 200 mg test substance/kg b.w./day.
Recovery period: Satellite animals: An increased salivation was still noted for 2 of 5 males previously treated with 200 mg test substance/kg b.w./day on test days 42 and 43. No test item related influence was noted at the end of the recovery period on test day 58.

HISTOPATHOLOGY:
The forestomach revealed a squamous cell hyperplasia with submucosal inflammatory reac-tion and hyperkeratosis/parakeratosis in the stra-tum corneum of the animals treated with 200 mg test substance/kg b.w./day. These lesions were completely reversible within the 16-days re-covery period. Further, a dose dependent increase of macro-phages with vacuolization in the mesenteric lymph nodes was observed in the animals treated with 100 or 200 mg test substance/kg b.w./day. The effect was still noted at the end of the recovery period.

Effect levels

Dose descriptor:

NOEL

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Histopathological changes noted in the forestomach and the mesenteric lymph nodes and signs of systemic toxicity (salivation) observed at dose levels of 100 and/or 200 mg/kg b.w./day, p.o.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The test substance was assessed for repeated dose toxicity according to OECD guidline 422. No test item-related mortality was noted. Starting at 100 mg test substance/kg b.w./day increased salivation was noted for very few male and female animals at 100 mg test substance/kg b.w./day and most animals at 200 mg test substance/kg b.w./day during all periods of the study (pre-mating, mating, post-mating, gestation and/or lactation period, respectively) starting on test day 1. Further, at 200 mg kg b.w./day laboured breathing was observed for one male and rough fur was noted several females during the pre-mating, mating, gestation and/or lactation period, respectively. Due to histopathological changes noted in the forestomach and the mesenteric lymph nodes and signs of systemic toxicity (salivation) observed at dose levels of 100 and/or 200 mg/kg b.w./day, p.o., the no-observed-effect level (NOEL) was 40 mg/kg b.w./day, p.o. via gavage for the F0 generation.

Executive summary:

In a guideline study (OECD 422) the test substance was administered by daily oral gavage to male and female Wistar rats at dose levels of 0, 40, 100 and 200 mg/kg body weight/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 31 or 36 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum,and at least 3 days of lactation (for 41 to 56 days).

No test item-related mortality was noted. Starting at 100 mg test substance/kg b.w./day increased salivation was noted for very few male and female animals at 100 mg test substance/kg b.w./day and most animals at 200 mg test substance/kg b.w./day during all periods of the study (pre-mating, mating, post-mating, gestation and/or lactation period, respectively) starting on test day 1. Further, at 200 mg kg b.w./day laboured breathing was observed for one male and rough fur was noted several females during the pre-mating, mating, gestation and/or lactation period, respectively. Due to histopathological changes noted in the forestomach and the mesenteric lymph nodes and signs of systemic toxicity (salivation) observed at dose levels of 100 and/or 200 mg/kg b.w./day, p.o., the no-observed-effect level (NOEL) was 40 mg/kg b.w./day, p.o. via gavage for the F0 generation.