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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 January 2010 to 15 October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully guidline and GLP compliant study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 3-Hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde,
dimethylolpropanale
- Trade name: Novaltan PF
- Hill formula: C5H10O3.
- Physical state: Colourless, high viscous
- Composition of test material, percentage of components: ca. 82% Dimethylpropanale, ca. 10% Hydroxymethanesulfonic acid, Na-salt (CAS No: 870-72-4), ca. 6% Water, ca. 2% Potassiumformiate (CAS No: 590-29-4).
- Lot/batch No.: CH 138067/001
- Expiration date of the lot/batch: 30 November 2010
- Stability under test conditions: min. 1 year
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
other: Crl: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, 97633 Sulzfeld, Germany
- Age at study initiation: About 8-9 weeks
- Weight at study initiation (first administration of the test substance: mean body weights between 400 and 406 g (males) and between 252 and 257 g (females)
- Fasting period before study: none
- Housing:
Dose Range Finding Study:
Makrolon cages Type III high version (39 cm x 23 cm ground area, 18 cm height).
Animals were double and single caged. Only animals of one group and one sex were caged together. Sanitation of cages once a week.

Main Study:
Single caging (in general, except for the mating period for and for dams with offspring).
Double caging (for the mating period, 1 male + 1 female per cage).
Group caging (1 dam plus her offspring per cage, for the post-partum period).
Makrolon cages Type III, high version (39 cm x 23 cm ground area, 18 cm height).
Sanitation of cages once a week.

- Diet (e.g. ad libitum):
Ssniff R/M-H maintenance diet for rats and mice (item V1534-3 ) ad libitum, supplied by Ssniff Spezialdiäten GmbH, 59494 Soest, Germany. Exception: Feed was withdrawn on days prior to blood sampling at 5:00 p.m., only from the animals, where blood was to be taken, and was re-offered immediately after the blood sampling.
Random samples of the feed are analysed for contaminants by the supplier. One sample is analysed also for contaminants in addition by an independent external laboratory. The limits of tolerance are derived from the "Deutsche Futtermittelverordnung" (German feed regulation).

- Water (e.g. ad libitum):
Tap water, acidified with HCl to pH >=3, from an automatic watering system, ad libitum. Random samples of the water are analysed by the "AGES", 1226 Vienna, Austria, to check, if the water fulfils the requirements for drinking water for humans (exception: the pH).

- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: Mean of temperature maxima: 20.27 °C. Mean of temperature minima: 20.38 °C.
- Humidity: Mean of humidity maxima: 49.40 %. Mean of humidity minima: 48.70 %.
- Air changes (per hr): 12
- Photoperiod: 12 (hrs dark / 12 hrs light)

IN-LIFE DATES:
Dose Range FInding Study: From: 20 January 2010 To: 28 January 2010
Main Study: From: 9 February 2010 To: 5 April 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Water: Water pro analysis, Merck item No. 1.16754.5000.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
An oral administration was performed by stomach intubations using a metal gavage once a day in the morning on 7 days per week.
The individual dose volumes were calculated using the last determined body weights.

VEHICLE
- Justification for use and choice of vehicle: The test substance sufficiently soluble in water.
- Concentration in vehicle: nominal 1, 31.6 and 100 g per L
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Preparation of vehicle: not appropriate
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation:until proven mating or until the end of the mating period of 14 days
- Proof of pregnancy: During the mating period, starting with the day after the commencement, all females were subjected to a daily examination (once in the morning,) for the presence of a vaginal plug. In case of absence of a vaginal plug, the females were subjected also once a day to a vaginal smear. The unstained vaginal smears were examined microscopically for the presence of sperm.
Presence of a vaginal plug or of sperm in the smears was taken as an evidence for successful mating.
"Day 0 of pregnancy" (of the given individual) was the day of proven mating.
"Day 0 post-partum" (of the given individual) was the day of birth (when parturition was complete).
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually-
- Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Determinations of the test substance in some selected preparations for concentration and homogeneity were performed. All details are given in a separate report, which is attached to the report.

Stability: Determined once prior to the study with a test substance preparation for the concentration of the low dosed group. A loss of 5% (10%) within 4 h at 10°C was tolerated, but was depending on the validation data of the analytical method.

Homogeneity: Not performed and not considered as necessary, as the test substance was administered as a solution.

Actual concentration: Determined in test samples prior to the study and once on Day 2. 3 samples of the preparations for groups A, B and C of 2.0 mL each were collected using syringe and stomach probe by the animal technician at the beginning, in the middle and at the end of the dosing procedure of the groups concerned. A deviation of the mean of the 3 samples from the target concentration of at most ± 10% was tolerated.
Duration of treatment / exposure:
"Day 1" (of the entire experiment) was the day of the 1st administration of the test substance. Commencement of dosing was at the beginning of the pre-mating period (see 2.6).
"Day 0 of pregnancy" (of the given individual) was the day of proven mating.
"Day 0 post-partum" (of the given individual) was the day of birth (when parturition was complete).
All adult animals were treated with the test substance solutions or with the vehicle once a day from Day 1 onwards until the day prior to their sacrifice (maximum duration: 54 days).
Frequency of treatment:
Once daily, 7 days/week
Details on study schedule:
not appropriate
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 316 and 1000 mg per kg body weight and day
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses chosen are derived from and based on the results of the Dose Range Finding Study.
Summarized results of the Dose Range Finding Study: All animals survived until their scheduled sacrifice. In all high dosed animals of both sexes unspecific signs of reduced well-being were noted in the last days of the dosing period. Body weights and - more pronounced - body weight gain of the high dosed males were reduced, when compared with the negative controls. No test substance related abnormalities were noted post mortem. No other indications for test substance related affects were found. None of the alterations observed is considered to constitute a severe toxic effect or to be life threatening.
In the Main Study, the high dose shall induce a clear toxicity, but no or at most isolated mortality. A dose of 1000 mg per kg body weight is commonly not exceeded as high dose. The low dose shall induce no toxic effect. The mid dose is interpolated geometrically.
Based on this, the doses of 100 mg and 316 mg and 1000 mg per kg body weight were selected for the Main Study in accordance with the study monitor.
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily, plus viability check once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to first test substance administration, then once a week, all adult animals.
Special emphasis was put on skin, fur, eyes, visible mucous membranes, incisors, secretion and excretion, body odour, autonomous activities (e.g. lacrimation, piloerection, pupillar size, abnormal breathing, and body surface temperature), vocalisation, abnormal locomotion, movements and posture, presence of convulsions or paralysis, stereotypes, bizarre behaviour, visible or palpable tissue masses.

BODY WEIGHT: Yes
- Time schedule for examinations:
Adult animals (except for pregnant females): Determined on Day 1, then once a week, and at termination.
Pregnant females: On Days 0, 7, 14 and 20 of pregnancy and within 24 h after parturition (i.e. Day 0 or 1 post partum) and on Day 4 post partum.
Offspring: The total weight of the litter is determined within 24 h after parturition (i.e. Day 0 or 1 post partum) and on Day 4 post partum.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes
Determined for weekly periods (except for changes in caging, e.g. at the beginning of mating or at proven mating; forming an interim endpoint), all animals (or per cage during mating period or with offspring).
- Mean daily diet consumption calculated as g food/kg body weight/day: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 0: The first 5 animals of each group. Day 15: The first 5 animals of each group.
- Anaesthetic used for blood collection: Yes (slight ether anaethesia)
- Animals fasted: Yes, overnight
- How many animals: Day 0: The first 5 animals of each group. Day 15: The first 5 animals of each group.
- Parameters examined:
Red blood cell count (RBC)
Haemoglobin concentration (HGB)
Haematocrit (HCT)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)
White blood cell count (WBC)
Mean cell volume (MCV)
Platelet count (PLT)
Differential white blood cell count (% of the different cell species)
Prothrombin time (Quick) as an indicator of blood clotting capacity.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 0: The first 5 animals of each group. Day 15: The first 5 animals of each group.
- Anaesthetic used for blood collection: Yes (slight ehter anaethesia)
- Animals fasted: Yes, overnight
- How many animals: Day 0: The first 5 animals of each group. Day 15: The first 5 animals of each group.
- Parameters examined:
Alanin aminotransferase (ALT, GPT)
Albumin
Alkaline phosphatase (AP)
Aspartate aminotransferase (AST, GOT)
Bile acids
Cholesterol
Creatinine
Gamma glutamyl transferase (GGT)
Glucose
Potassium (K+)
Sodium (Na+)
Total protein
Urea

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Males: Once in the last week of the dosing period.
Females: Once during the lactation period (i.e. on Days 1-4 post partum).
Offspring: Not examined.
- Dose groups that were examined: all
- Battery of functions tested: Assessment of the behaviour, the motor activities, and the sensory reactivity to different stimuli (acoustic, tactile, visual and proprioceptive) and grip strength.

OTHER:

Sacrifice and pathology
GROSS PATHOLOGY: Yes
Adult animals were killed by inhalation of 80 % CO2 plus 20 % air and subjected to a necropsy including a gross pathological examination immediately after death on
Males: On the day after the end of the mating period.
Females: On Day 4 post partum or Day 55 (i.e. 26 days after the end of the mating period).

The following organs/tissues (if appropriate) were fixed in 4 % buffered formaldehyde, with the exception of the eyes (fixed in Davidson's fixative) and the skin and the testes (both fixed in Bouin's solution, the testes were punctured on both poles with a needle before immersion in the fixative).

gross lesions, tissue masses or tumours
adrenal glands
brain including cerebrum, cerebellum and pons)
coagulating glands
epididymides
eyes
heart
kidneys
large intestine (colon)
liver
lungs
lymph nodes (mandibular, mesenteric)
ovaries
prostate
sciatic nerve
seminal vesicles
skeletal muscle (thigh)
small intestine (duodenum, ileum, jejunum; including Peyer's patches), prepared as "Swiss Roll"
spinal cord (cervical, thoracal, lumbar)
spleen
sternum with bone marrow
stomach
testes
thymus
thyroid glands
trachea.
urinary bladder
uterus
vagina with cervix

Additional examinations were performed in the females:
Counting of corpora lutea per ovary (visual).
Counting of implantation sites (visual, no staining).

The pups were killed by overdosed chloroform anaesthesia at the same time as their mothers and subjected to an external examination for gross abnormalities. No organs are weighed or preserved in the pups.

ORGAN WEIGHTS: Yes
Fresh weights of the following organs were determined of all adult animals at necropsy:
adrenal glands (both together)
brain
epididymides (both together)
heart
liver
kidneys (both together)
prostate and seminal vesicles with coagulating glands
spleen
testes (both together)
thymus
Relative organ weights were calculated by relating the absolute organ weights to the last determined individual body weight and to the brain weight.

HISTOPATHOLOGY: Yes
Groups K and C:
Histopathological examination was performed in the first 5 adult animals of groups K and C, of all fixed organs and tissues listed above.
Groups A and B:
Liver: first 5 males and first 5 female animals.
Stomach: first 5 males.
Justification: Indication for test substance related effects in group C.
The tissue trimming was performed according to "Bahnemann et al.: RITA - Registry of Industrial Toxicology Animal Data - Guides for Organ Sampling and Trimming Procedures in Rats"; Exp.Toxicol.Pathol. 47 (1995), p 247 ff. with the following exceptions:
Not all possible sections, as given in the literature, were actually prepared. One section per organ (in paired organs one of each) was made with the following exceptions:
Brain (3 sections, one at the optic chiasma, the second at the caudal border of the mammillary body, just posterior to the attachment of the pituitary and the third about 2 mm caudal to the transverse fibres of the pons). Representative regions of the brain, especially cerebrum, cerebellum and pons were included in these sections.
Spinal cord (three sections, a cervical, a thoracal and a lumbar).
Liver (two sections).
The small intestine (duodenum, jejunum and ileum) was fixed and trimmed to form two "Swiss Rolls" (one of the cranial and one of the caudal part), according to "Moolenbeek and Ruitenberg: The Swiss Roll, a simple technique for histological studies of rodent intestine"; Lab. Animals 15 (1981), p.57-59.
The trimmed samples of organs or tissues, as described above, were embedded in paraffin. Sections of about 5 µm were stained with haematoxylin and eosin (H&E). Evaluation of slides was performed using a light microscope Leica-DMRB.
To describe the severity of lesions, the following grades were applied, if appropriate:
minimal (1), mild (2), moderate (3), marked (4), severe (5).
The term "focal" together with a higher degree of severity also stands for "multifocal".
Oestrous cyclicity (parental animals):
not recorded
Sperm parameters (parental animals):
not applicable
Litter observations:
STANDARDISATION OF LITTERS
not appropriate

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
• Runts: Pups, that are substantially smaller than corresponding control pups.
• Abnormal pups: Pups showing any abnormalities or malformations.
• Sex ratio: Number of males / number of females.
• Litter weight: Sum of the body weights of all live pups.
• Pup weight: Litter weight / number of pups.
• Pre-natal, post-implantation loss of offspring: Implants minus live births.
• Post-natal loss of offspring: Live births minus pups alive on Day 4 post partum.


GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
see above (Parental animals: Observations and examinations)
Postmortem examinations (offspring):
none performed
Statistics:
Analysis of variance followed by the Scheffé-test: all data with means and standard deviations determined, comparison of more than two groups
t-test: all data with means and standard deviations determined, for comparison of two groups only
H-test of Kruskal and Wallis followed by the test of Nemenyi: counted events with scoring or in cases where the requirements for the analysis of variance were not fulfilled
Chi2-test: counted events
Fisher's exact test: counted events, if the Chi2-Test was not applicable

Results were analysed separately for males and females. P = 0.05 was chosen in each test. Two tailed test were used.
Numerical data have been rounded for presentation; a manual recalculation therefore may yield slightly different results to those given in the tables.
Please note: Whenever the term "significant" is used in this report, it stands for "statistically significant".
Reproductive indices:
• Pairs started: Number of pairs at the beginning of the mating period.
• Females showing evidence of copulation: Females with a vaginal plug or with sperm in the vaginal smear.
• Females achieving pregnancy: Number of females giving birth to live or dead pups.
• Duration of pregnancy: Days between proven mating and parturition.
• Corpora lutea per dam: Sum of corpora lutea of both ovaries.
• Implants per dam: Number of implantation sites in the uterus, found at necropsy on Day 4 post partum.
Offspring viability indices:
Runts: Pups, that are substantially smaller than corresponding control pups.
Abnormal pups: Pups showing any abnormalities or malformations.
Sex ratio: Number of males / number of females.
Litter weight: Sum of the body weights of all live pups.
Pup weight: Litter weight / number of pups.
Pre-implantation loss of offspring: Corpora lutea minus implants.
Pre-natal, post-implantation loss of offspring: Implants minus live births.
Post-natal loss of offspring: Live births minus pups alive on Day 4 post partum

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY
2 animals died in the course of blood sampling on Day 0 and were replaced by spare
animals. All other animals survived until their scheduled sacrifice.

OBSERVATIONS IN LIFE
Alopecia which is known as a spontaneous alteration of the strain of rats used, was noted in several individuals. Its presence is not related to the test substance. One animal from the mid-dosed group (No. 23) showed a tissue mass on the upper lip on days 11 to 27. At the time of necropsy the tissue mass was not detectable anymore.
No additional information could be derived from the detailed observations.

FUNCTIONAL OBSERVATIONS AND GRIP STRENGTH DETERMINATION
No test substance related findings were made nor were there any significant group differences at the functional observations. All results represent a normal pattern of behaviour and normal reactions of rats of the strain and age examined.
There was no significant group difference in the grip strengths of both sexes.

BODY WEIGHTS AND BODY WEIGHT GAIN
No significant difference in body weight and body weight gain was noted between the groups
In the body weights of the females before mating and of the non-inseminated dams no significant differences were found.

FEED CONSUMPTION
There were no noteworthy differences or dose related trends noted in the feed consumption of both sexes.

HEAMATOLOGY
There were no significant differences nor dose related trends in the haematological parameters of both blood sampling terms, on Day 0 and on Day 15.

CLINICAL BIOCHCEMISTRY
Elevated ALT levels (Day 15) in the mid dosed males are not given toxicological relevance, as there was no dose-response relationship.

GROSS PATHOLOGY
No findings were made at the gross examination at necropsy which gave an indication for a specific mode of action of the test substance. At the time of necropsy of animal number 23 there was no abnormal tissue mass detectable.

ORGAN WEIGHTS
No significant differences in organ weights between the groups could be found.

HISTOPATHOLOGY
Histopathologically, statistically significant indications for a slight gastric irritation (proliferation of the limiting ridge) without signs of inflammation were noted in some male animals of the high dosed group. This finding is interpreted as a slight local irritation by the test substance and is not given major toxicological relevance.

In the livers a slight centrolobular hepatocyte cytoplasmatic basophilia was noted statistically significant in male animals of the high dosed group. This is interpreted as an indicator of enzyme induction. This finding is taken as an adaptive response and not a toxic effect.

Extramedullary haematopoiesis in the spleens of the high dosed females is a sequel of the blood loss at birth, 4 days before sacrifice.

Some other isolated findings in histopathology are regarded as part of the background pathology, inconspicuous in type and incidence. None of them is related to the test substance.


MATING PERFORMANCE
In 2 mid dosed females no indications for successful mating (vaginal plug or presence of sperm) were found. Nevertheless the animals became pregnant and gave birth.
No indication for an effect of the test substance on mating performance was found.



PREGNANCY, BIRTH, PERI- AND POSTNATAL PERIOD
One pregnant female (no.123, group B) was found with reddish-brown bedding material in the cage. Subsequently the animal showed no signs of pregnancy anymore. At necropsy corpora lutea and two implantation sites were found. Therefore it was supposed that the animal cannibalised its offspring after unwittnessed birth.
This single case is not related to the test substance.
Again, no test substance related group differences were noted.

OFFSPRING
There was no indication for a test substance related effect on the offspring.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
316 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
870 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No toxic effectsl were noted at the high dose in females. The actual high dose was 870 mg per kg body weight and day.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

BIRTH, PERI- AND POSTNATAL PERIOD

One pregnant female (no.123, group B) was found with reddish-brown bedding material in the cage. Subsequently the animal showed no signs of pregnancy anymore. At necropsy corpora lutea and two implantation sites were found. Therefore it was supposed that the animal cannibalised its offspring after unwittnessed birth.
This single case is not related to the test substance.
Again, no test substance related group differences were noted.

OFFSPRING

There was no indication for any test substance related effect on the offspring.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
870 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mating index; fertility index; % females mating > 1st estrous; number of implantation sites; duration of pregnancy; birth index; live birth index; pregnancy index; litter size; litter weight; pup weight; sex ratio; survival index; viability index;

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The No-observed-effect-level (NOEL) of "3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde" was at 870 mg per kg body weight for females and 316 mg per kg body weight for males.
According to EU-Directive 2001/59/EC, a classification with "R48" is not considered as necessary for "3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde", as no severe toxic effects were noted at doses of up to 870 mg/kg.
According to EU-Directive 2001/59/EC, a classification with "R60" is not considered as necessary for "3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde", as no toxic effects on reproduction were noted.
According to Regulation (EC) No. 1272/2008 (CLP), "3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde" needs not to be classified for "Reproductive Toxicity" and also not to be classified for "Specific Target Organ Toxicity after Repeated Exposure".
Executive summary:

Aim of the study

The study was performed to assess and evaluate the toxic characteristics of the test substance, resulting from a subacute oral administration via gavage to rats and comprises a reproduction and developmental toxicity screening test, according to OECD-Guideline 422,. A Dose Range Finding Study preceded this study.

Methods

All details given here refer to the Main Study; a survey of the methods of the Dose Range Finding Study is included into the Results of the Dose Range Finding Study (see below).

 

Test substance:"3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde". 

Test substance preparation for administration:Freshly dissolved with the vehicle.

Vehicle:Water.

Route of test substance administration:Oral via gavage.

Dosing regimen:1/day for 28 consecutive days in males. 1/day until Day 4 post partum or for 55 Days in females. No test substance administration in the offspring.

Dose volume:10 mL test substance preparation or vehicle per kg body weight.

Test system (animals):Rats, Crl:CD(SD). 10 males and 10 females per group.

Groups, doses:

K (negative control group)                vehicle only,

 A (low dose group)                                        100 mg per kg body weight,

B (mid dose group)                                       316 mg per kg body weight,

C (high dose group)                                     1000 mg per kg body weight,

The target doses are derived from and based on the results of the dose range finding study.

For a survey thereof, see below.

Experimental schedule:

2 weeks pre-mating period;

2 weeks mating period; then sacrifice of the males;

maintenance of the successfully mated females throughout their pregnancy, birth, until Day 4 post partum, then sacrifice of the dams and their offspring;

maintenance of the non-successfully mated females for 26 days after the end of the mating period.

"Day 1"(of the entire experiment) was the day of the 1stadministration of the test substance.

"Day 0 of pregnancy"(of the given individual) was the day of proven mating.

"Day 0 post-partum"(of the given individual) was the day of birth.

 

Investigations:

Analyses of the test substance preparations: In selected samples. For homogeneity and concentration.

Animal observations: All animals, once a day, plus a daily check for viability.

Detailed clinical observations: All parental animals, once a week.

Functional observations: All parental animals, once, prior to sacrifice.

Body weights: All males and all females prior to successful mating once a week. All successfully mated females on Days 0, 7, 14, 20 of pregnancy and on Days 0 and 4 post partum. Pups as total litter weight on Days 0 and 4 post partum

 Feed consumption: All animals, for weekly periods.

Haematology: The first 5 parental animals of all groups, prior to first dosing (Day 0) and on Day 15.

Clinical biochemistry: The first 5 parental animals of all groups, prior to first dosing (Day 0) and on Day 15.

Necropsy with gross pathological examination:All males on Day 15, all successfully mated females on Day 4 post partum, and all non-successfully mated females on Day 55. No necropsy in pups.

 Organ weight determination: Selected organs in all parental animals at necropsy. Not determined in pups.

Histopathological examination: Selected organs or tissues in the first 5 parental animals of groups K and C. Organs and tissues with suspected test substance related alterations also in groups A and B.
No histopathology in pups.

Data on reproduction and developmental performance: Results of vaginal smear examinations; duration of pregnancy; number, sex and viability of offspring. Number of corpora lutea and implantation sites in the dams. Several parameters were additionally derived from the primary data above.

Results

Dose Finding Study:

Doses of 100 mg or 316 mg or 1000 mg per kg body weight were given to groups of 5 male and 5 female rats once a day for 7 consecutive days. Investigations performed: Animal observations, body weights, feed consumption, and gross examination at terminal necropsy. Summarised results: All animals survived until their scheduled termination and were found to be normal at the daily observations. Body weights, body weight gain, feed consumption did not differ significantly or notably between control and test substance exposed animals. All animals were normal at gross examination during the terminal necropsy.

Main Study:

Analyses of the test substance preparations: The test substance was found to be sufficiently stable in the preparations.The concentrations of the test substance in the preparations for groups A and B were within the chosen limits, while the determined concentration for group C was found to be clearly too low (87 % of the target concentration). As a consequence of this, the actual dose for the high dose group is defined at 870 mg per kg instead of 1000 mg per kg, the target dose.A fraction of the test substance was missing in the final preparations, possibly by being placed during the weighing procedure on positions of the beaker without contact to the vehicle and thus not being dissolved. This might have happened due to the high viscosity of the test substance and low absolute amounts weighed in. As a consequence, the actually administered doses were, at least on some terms of the administration period, lower than the target doses. For the estimation of the actually administered doses, the analytical data with the highest deviation from the target were used.

Mortality 2 animals died in the course of taking blood samples on day 0 and were replaced by spare animals.

 Observations in life, clinical and functional observations, grip strength determination: Loss of hair was observed in several animals in all dosed groups.

Body weights and feed consumption: There were no significant differences in body weights between the groups.

Haematology: No significant group differences were noted at both blood sampling terms.

Clinical biochemistry: Only, but all, parameters with significant differences to the negative control group (indicated by a black background) are given in this survey:

parameter (sex)

low dose
100 mg/kg

(% of the negative controls)

mid dose
316 mg/kg

(% of the negative controls)

high dose
1000 mg/kg

(% of the negative controls)

glucose (males)

99

87

95

The significant group differences are not given a toxicological relevance.

  Day 15:

parameter (sex)

low dose
100 mg/kg

(% of the negative controls)

mid dose
316 mg/kg

(% of the negative controls)

high dose
1000 mg/kg

(% of the negative controls)

ALT (males)

108

116

101

The elevated ALT level in the mid dosed group is not given a toxicological relevance due to the lack of a dose response.

 

Organ weight determination: There were no significant group differences in organ weights.

Necropsy with gross pathological examination and histopathology: Histopathologically, indications for a slight gastric irritation (proliferation of the limiting ridge) without signs of inflammation were noted in some high dosed males. A slight basophilia of centrolobular hepatocytes is interpreted as a possible indication of enzyme induction. This alteration is taken as an adaptive response and was present statistically significantly only in high dosed males.

Reproduction data: No indications for a test substance related effect was made with any of the reproduction parameters.

Discussion and Conclusion

The test substance caused minimal irritation on the mucous membranes of the stomach noted by a proliferation of the limiting ridge in male animals of the high dose group.

 

In the livers of the high dosed males a centrolobular cytoplasmatic basophilia of the hepatocytes is interpreted as indicator of an induction of metabolizing enzymes. As the liver weight was not affected, the amount of enzyme induction is assumed to be rather low. Enzyme induction is not taken as an adverse effect, but as an adaptive response.

 

The few other alterations were not given toxicological significance.

There was no indication noted for a systemic toxic effect of the test substance.

There was no indication for an effect of the test substance on the reproduction.

There was no pronounced sex difference in the response to the test substance.

The effects noted histopathologically were minimal to mild.

 

The determined concentration of the test substance in group C was found to be approximately 870 mg/kg and therefore less than the target concentration of 1000 mg/kg. Therefore the NOEL for the female animals had to be corrected from 1000 mg/kg (target concentration) to 870 mg/kg (actual determined concentration).

 

The No-observed-effect-level (NOEL) and the No-observed-adverse-effect-level (NOAEL) of "3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde" was at 870 mg per kg body weight for females and 316 mg per kg body weight for males.

 

According to EU-Directive 2001/59/EC, a classification with "R48" is not considered as necessary for "3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde", as no severe toxic effects were noted at doses of up to 870 mg/kg.

 

According to EU-Directive 2001/59/EC, a classification with "R60" is not considered as necessary for "3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde", as no toxic effects on reproduction were noted at doses of up to 870 mg/kg body weight.

 

According to Regulation (EC) No. 1272/2008 (CLP), "3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde" needsnotto be classified for "Reproductive Toxicity" and also not to be classified for "Specific Target Organ Toxicity after Repeated Exposure".