acetalization product between glucose and C16-18(even numbered)- alcohol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Acetalization product between glucose and C16/18 (even numbered)-alcohol was administered in Wistar rats by gavage for 90 consecutive days with a 28-days recovery period, according to an OECD test n°408. Detailed testicular histopathological examination was conducted in order to identify possible treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen. Intact epididymis was also examined to include the caput, corpus, and cauda, which was accomplished by evaluation of a longitudinal section. The epididymis was evaluated for possible leukocyte infiltration, change in prevalence of cell types, aberrant cell types, and phagocytosis of sperm; As described in paragraphs 41 to 44 (OECD n°416). The repeated administration of test item did not cause any toxicological effects on general health, mortality, functional observational battery, growth and food consumption. No test item-related changes observed in haematology, coagulation, clinical chemistry, urinalysis, terminal fasting body weights, organ weights/ratios and sperm parameters and gross and histopathological changes. Hence, the evaluated NOAEL is 1000 mg/kg Body weight/day under the test conditions and doses employed.

Furthermore, several reproduction toxicity studies are available on read-across and source substances and did not show any adverse effects linked to the tested substances: 2 screening for reproductive / developmental toxicity studies (OECD 422) performed with two members of the category (acetalization product between glucose and C14 alcohol and acetalization product between glucose and C20/22 alcohol) concluded to a NOAEL value for toxicity to reproduction of 1000 mg/kg for P0 and F1.

In a reliable study (Iglesias 2002) conducted according to a protocol similar to OECD guideline 415 (ICH S5(R2)) and performed with the source substance (docosan-1-ol), a NOAEL of 1000 mg/kg bw/day in the rat was determined for the reproductive effects. Another study, a 26 weeks toxicity study also conducted with docosan-1-ol in the rat by oral route, did not show any adverse effect on reproductive organs at 1000 mg/kg. Moreover an OECD test guideline n°421 done with another source substance, Alkyl polyglucosides C10-16, showed that no adverse effects were observed regarding male and female reproductive organs even at the highest dose of 1000 mg/kg bw/day, in the rats. So based on the OECD test 408 with parts of 416 on the registered substance, 2 toxicity studies (OECD 422) performed with 2 category members and 3 reproductive toxicity studies conducted with source substances (C22 alcohol and C10-16 APG), there is no need to perform a two generation study (or one-extended generation study) with the registered substance.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

22 April 2008 - 22 june 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions. Read across from a substance of the category acetalization product between glucose and C20/22(even numbered)-alcohol

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

except during study days -6 to 9, and except for the absence of chemical analyses of dosage forms

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder
- Age at first treatment: approx. 10 weeks
- Weight at first treatment (mean): M=433 g, F=276 g
- Housing: individually, except during pairing
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Relative humidity: 50 ± 20%
- Light/dark cycle: 12h/12h
- Ventilation: 12 cycles/hour of filtered, non-recycled air.

IN-LIFE DATES: beginning: 29 April 2008 / end: up to 21 June 2008

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
test item was ground, heated to 80°C, mixed with vehicle heated to 80°C, forming a suspension.
The test item dosage forms were prepared daily

VEHICLE
- Justification for use and choice of vehicle (if other than water): lipophilicity of the substance
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/day
- Lot/batch no. (if required): 057K6093, A0247283 and 1223873
- Purity: not indicated

Details on mating procedure:

- M/F ratio per cage: 1
- Length of cohabitation: until mating occurred
- Proof of pregnancy: vaginal plug, or sperm in vaginal smear - referred to as day 0 post-coitum
- In case of unsuccessful pairing: not detailed (pairing always succeeded)
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Absence of a practical method of analysis

Duration of treatment / exposure:

from 2 weeks before mating until the end of mating (males: total of 39 days) or day 5 pp (females: total of 44-55 days)

Frequency of treatment:

once daily

Details on study schedule:

- no F1 parents (only one generation mated).
- Age at mating of the mated animals in the study: 13-16 weeks

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day (m/f)
Basis:
other: nominal per gavage

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no serious effects at up to 1000 mg/kg/day in a 14-day range-finding study, see 7.5.1
- Rationale for animal assignment: computerized stratification procedure (average body weight of each group is similar)

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule: day 1 and then weekly; and in F also days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum

FOOD CONSUMPTION for each animal: Yes
- Time schedule: weekly (last 7-day consumption period)

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

Yes : from a fresh vaginal lavage, each morning during the mating period, until the females were mated

Sperm parameters (parental animals):

No seminology examinations.
Testis and epididymis : weight (all males), microscopic exmaination (some rats: see table 1).

Litter observations:

STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight on days 1 and 5, clinical signs

Postmortem examinations (parental animals):

SACRIFICE
- All male survivors: after the end of the mating period
- All female survivors: on day 6 post-partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- Sacrifice on non-selected breeders/progeny: not applicable (only 1 generation of parents and of offspring)
- All pups sacrificed on day 5 post-partum

GROSS NECROPSY: Yes, on pups sacrificed and found dead
- external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS: Not performed

Reproductive indices:

Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites
Mating index = 100 * Number of mated animals / Number of paired animals
Fertility index = 100 * Number of pregnant female partners / Number of mated pairs
Gestation index = 100 * Number of females with live born pups / Number of pregnant females
Live birth index = 100 * Number of live born pups / Number of delivered pups

Offspring viability indices:

Viability index = 100 * Number of surviving pups on day 5 post-partum / Number of live born pups

Clinical signs:

effects observed, treatment-related

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

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

The only clinical signs observed were hypersalivation and reflux at dosing, which were considered to be related to treatment with the test item but were non-adverse.

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: NOAEL = highest tested dose, without relevant effects.

Dose descriptor:

NOEL

Remarks:

reproduction (mating and fertility)

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: NOEL = highest tested dose, without relevant effects.

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

Not required (no effects)

Dose descriptor:

NOEL

Remarks:

offspring toxicity

Generation:

F1

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: NOEL = highest tested dose, without relevant effects.

Reproductive effects observed:

not specified

Not required (no effects)

Conclusions:

Under on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day, the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day and the NOEL for toxic effects on progeny was 1000 mg/kg/day.
Considering REACH Annex IX 8.7.2 and 8.7.3 criteria alone, a developmental toxicity study and a two-generation study in one species should be proposed as the substance is not genotoxic or toxic to reproduction according to available data, and there are no data on plasmatic exposure after dosing. However, although no exemption can be derived from these criteria, it should be underlined that it seems irrelevant to propose and perform this test, both from scientific and regulatory standpoints:
- the substance is clearly non-toxic based on the available tests including evaluations of fertility and development (as detailed above),
- it is expected to be metabolised into endogenous compounds (as discussed under 5.1),
- the expected metabolism and excretion do not suggest any bioaccumulation potential (as discussed under 5.1),
- exposure of workers is negligible when taking into account the high particle size limiting dermal and respiratory absorption (as discussed under 5.1), and the various risk management measures which are applied by SEPPIC (summarized under part A.1),
- the risk assessment for consumers of the final cosmetic product is exempted under REACH, Directive 2003/15/EC forbids in vivo testing on cosmetic ingredients for the dossier of the final cosmetic product from March 2009, and cosmetic regulations are stated to prevail over REACH requirements.

Executive summary:

The test item, LCE07104, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, gestation and until day 5 post-partum, at dose-levels of 100, 300 or 1000 mg/kg/day.

There were no unscheduled deaths during the study and the only clinical signs observed were hypersalivation and reflux at dosing, which were considered to be related to treatment with the test item but were non-adverse. There were no treatment-related effects on body weight, body weight gain or food consumption at any dose-level. There were no differences from controls for pairing, mating and fertility parameters. Pups showed no effects of treatment on survival or body weight performance.

There were no treatment-related effects on organ weights and no treatment-related macroscopic or microscopic findings were observed.

Considering REACH Annex IX 8.7.2 and 8.7.3 criteria alone, a developmental toxicity study and a two-generation study in one species should be proposed as the substance is not genotoxic or toxic to reproduction according to available data, and there are no data on plasmatic exposure after dosing. However, although no exemption can be derived from these criteria, it should be underlined that it seems irrelevant to propose and perform this test, both from scientific and regulatory standpoints:

-     the substance is clearly non-toxic based on the available tests including evaluations of fertility and development (as detailed above),

-     it is expected to be metabolised into endogenous compounds (as discussed under 5.1),

-     the expected metabolism and excretion do not suggest any bioaccumulation potential (as discussed under 5.1),

-     exposure of workers is negligible when taking into account the high particle size limiting dermal and respiratory absorption (as discussed under 5.1), and the various risk management measures which are applied by SEPPIC (summarized under part A.1),

the risk assessment for consumers of the final cosmetic product is exempted under REACH, Directive 2003/15/EC forbids in vivo testing on cosmetic ingredients for the dossier of the final cosmetic product from March 2009, and cosmetic regulations are stated to prevail over REACH requirements.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

from 2009-05-07 to 2009-12-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study has been performed in compliance with the: Swiss Ordinance relating to Good Laboratory Practice adopted May 18th, 2005 [SR 813.112.1]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted on November 26th, 1997 by decision of the OECD Council [C (97)186/Final]. These principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), and Japan (MHLW, MAFF and METI). There were no circumstances that may have affected the quality or integrity of the data.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

TYPES OF QA INSPECTIONS: study plan; study based: test item, test system, RD, dose preparation, sample taking, treatment, BW, FOB, necropsy; process based: clinical laboratory investigations, histotechnique, analytical work up; analytical appendix; report

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Animals: Rat, HanRcc: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories Ltd., Laboratory Animal Services, Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
Number of Animals: 40 males (10 per group) and 40 females (10 per group)
Age (at Start of Treatment): 11 weeks
Body Weight Range (at Start of Treatment): Males (286 to 327 g) and females (178 to 221 g)
Identification: Cage card and individual animal number (ear tattoo). Pups: On day 1 post partum, pups were individually tattooed with Indian ink.
Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually following room cleaning, and are considered not to have any influence on the study. Therefore, these data are not reported but are retained at Harlan Laboratories. There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier&Söhne GmbH&CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
Diet: Pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no. 76/08).
Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

DOSE FORMULATIONS

The dose formulations were prepared daily using the test item as supplied by the Sponsor.

LCE08129 had to be crushed in order to get small portions for weighing. It was weighed into a glass beaker on a tared precision balance, heated in a water bath (max. 70 °C) and the vehicle was added (w/v) under agitation. Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

TREATMENT

Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
Frequency of Administration: Daily
Target Dose Levels: Group 1: 0 mg/kg/day (control group); Group 2: 100 mg/kg/day; Group 3: 300 mg/kg/day; Group 4: 1000 mg/kg/day
Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range finding toxicity study in Han Wistar Rats, Harlan Laboratories Study C23966, using dose levels of 100, 300 and 1000 mg/kg/day, resulting in a NOEL of 1000 mg/kg/day.
Dose Volume: 5 mL/kg body weight
Duration of Acclimatization Period: 7 days
Duration of Treatment Period: Males (28 days); females (approximately 7 weeks)

Details on mating procedure:

MATING, GESTATION AND LACTATION

During the pairing period, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:

- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.

The day of mating was designated day 0 post coitum.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DOSE FORMULATIONS

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (4 hrs at room temperature). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Analytic Department (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by HPLC coupled to an UV detector following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

The application formulations investigated during the study were found to comprise LCE08129 in the range of 74.0% to 108.6%. Eleven out of 12 samples met the required content limit of ±20% with reference to the nominal concentration. The homogeneous distribution of LCE08129 in the preparations was approved because single results found did not deviate more than 3.3% (<15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept 4 hours at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

Duration of treatment / exposure:

Males: 28 days
Females: Approximately 7 weeks

Frequency of treatment:

Daily

Details on study schedule:

Acclimatization: 7 days minimum (males and females)
First Test Item Administration: Day 1 of pre-pairing (males and females)
Pre-Pairing: 14 days (males and females)
Pairing: 14 days maximum (males and females)
Gestation: Approximately 21 days (females)
Treatment Ends: On day 4 post partum (females); on day before sacrifice (males)
Necropsy: On day 5 post partum (females); on day 4 post partum (pups); after treatment of at least 28 days, when no longer needed for assessment of repro-ductive effects (males)

Remarks:

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

Remarks:

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

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
Food Consumption: Males (weekly during pre-pairing and after pairing periods); females (pre-pairing period days 1-8 and 8-14; gestation days 0-7, 7-14 and 14-21 post coitum, and days 1-4 post partum). No food consumption was recorded during the pairing period.
Body Weights: Recorded daily from treatment start to day of necropsy.


DETAILED CLINICAL OBSERVATIONS
Once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside the home cage. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.


FUNCTIONAL OBSERVATION BATERY
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were performed with five P generation males and five P generation females from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:

- Cage-side observations: unusual body movements (e.g. tremors, convulsions), abnormal behavior (e.g. circling, stereotypy) and posture as well as resistance to removal.

- Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex and reaction to handling.

- Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.

- Categorical observations (can be made any time during the FOB): hair coat, behavior, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or feces, soiling, general abnormalities, posture.

- Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

Any abnormal findings were recorded and, where appropriate, graded in severity.

Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.


CLINICAL LABORATORY INVESTIGATIONS
Blood samples were obtained on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

The assay was performed at Clinical Diagnostics Department (Harlan Laboratories Ltd., Füllinsdorf) under internal laboratory quality control conditions to assure reliable test results.


HEMATOLOGY
The following hematology parameters were determined:

Complete Blood Cell Count:
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Leukocyte count, total
Differential leukocyte count
Platelet count

Coagulation:
Prothrombin time (= Thromboplastin time)
Activated partial Thromboplastin time


CLINICAL BIOCHEMISTRY
The following clinical biochemistry parameters were determined:
Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Aspartate aminotransferase
Alanine aminotransferase
Alkaline phosphatase
Gamma-glutamyl-transferase
Bile acids
Creatine kinase
Sodium
Potassium
Chloride
Calcium
Phosphorus
Protein, total
Albumin
Globulin
Albumin/Globulin ratio

Litter observations:

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

Postmortem examinations (parental animals):

TERMINATION OF THE STUDY

Males were sacrificed after treatment of 28 days. Dams were sacrificed on day 5 post partum.

If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

NECROPSY

All parent animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

All parent animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
For the parent animals, special attention was directed at the organs of the reproductive system.

The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS

The testes and epididymides of all parental males were weighed as pairs.

In addition, from 5 males and females killed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken:
Adrenal glands (weighed as pairs)
Brain
Heart
Kidneys (weighed as pairs)
Liver
Thymus
Spleen

TISSUE PRESERVATION

The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
Prostate
Seminal vesicles with coagulating gland
Testes (in Bouin’s fixative)
Epididymides (in Bouin’s fixative)

The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
Ovaries

In addition, from the five males and females per group selected for organ weights and from all animals found dead or killed in extremis, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
Gross lesions
Brain
Spinal chord
Small and large intestines (incl. Peyer’s patches)
Stomach
Liver
Kidneys
Adrenals
Spleen
Heart
Thymus
Thyroids, and parathyroids if possible
Trachea and lungs (preserved by inflation with fixative and then immersion)
Uterus (with vagina)
Urinary bladder
Lymph nodes (mesenterial, mandibular)
Peripheral nerve (sciatic)
Bone marrow

HISTOTECHNIQUE

All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the study pathologist.

HISTOPATHOLOGY

Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.

Postmortem examinations (offspring):

TERMINATION OF THE STUDY

Pups were sacrificed on day 4 post partum.

NECROPSY

All pups sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

All pups were sacrificed by an injection of sodium pentobarbital.

All pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

Statistics:

Following statistical methods were used to analyze locomotor activity, food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables coulc be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses and mean litter size.

Offspring viability indices:

From the on-line recorded reproduction data, the following parameters were calculated: dead/live pups at first litter check, pup sex ratios and postnatal loss (up to day 4 post partum).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At the dose levels of 300 and 1000 mg/kg bw/day all males and females pushed their head through the bedding material and showed salivation after application. These findings were considered to be signs of discomfort but not a toxic effect.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At the dose level of 1000 mg/kg bw/day males mean food consumption and mean body weight gain were slightly reduced after treatment start . This transient reduction was considered to be test item-related but not adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At the dose level of 1000 mg/kg bw/day males mean food consumption and mean body weight gain were slightly reduced after treatment start . This transient reduction was considered to be test item-related but not adverse.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

As examined by testis and epididymis weight.

Reproductive performance:

no effects observed

1 IN-LIVE DATA - PARENTAL ANIMALS
1.1 DETAILED CLINICAL OBSERVATIONS (DAILY)
(See Summary Tables on pp. 22-23)

In group 3 and 4, all animals pushed their head through the bedding material and showed salivation after application during the whole treatment period. These findings were considered to be a sign of discomfort but not a toxic effect.

In group 3 and 4, one and two males, respectively, had rales on single days during treatment. Furthermore in group 4, one male which showed rales had also ruffled fur. These findings were considered to be not test item-related.

1.2 DETAILED CLINICAL OBSERVATIONS (WEEKLY)
No test item-related findings were noted during the detailed weekly clinical observations of males and females in any group.

1.3 FUNCTIONAL OBSERVATIONAL BATTERY
(See Summary Tables on pp. 24-27; A description of the test parameters and scoring used on pp. 28-32)

None of the parameters under investigation during the functional observational battery (appearance, behavior in the open field, grip strength, landing food splay, body temperature) gave an indication of a test item-related effect.

In group 4, male no. 35 in group 4 was aggressive, had unkempt and ruffled fur and vocalization over 10 seconds. Due to the isolated occurrence, this behavior was considered to be incidental. Furthermore, for three males the number of rearings was decreased compared to the control group. Since the locomotor measurements did not show any reduced activity, this finding was considered to be incidental. For two females, a wet chin was noted due to the observed salivations.

Also in group 1, 2 and 3, single animals were noted, which showed an aggressive behavior, a reduced number of rearings or a spontaneous vocalization.

1.4 LOCOMOTOR ACTIVITY
Locomotor activity was assessed quantitatively in terms of low beam counts in activity monitor.

No indication of a test item-related effect was noted in any group.

1.5 FOOD CONSUMPTION - MALES
(See Figures on pp. 2-3, Summary Tables on pp. 33-34 and 38-39)

Pre-pairing and After Pairing Periods

In group 4, mean food consumption was transiently statistically significantly reduced after treatment start ( 15.3% compared to the control group from day 1 - 8 of the pre-pairing period). Thereafter, mean food consumption was not affected by treatment with the test item. This transient reduction was considered to be test item-related but not adverse.

In group 3, mean food consumption was lower in the second week of the pre-pairing period ( 9.1% compared to the control group). Due to the absence of a dose dependency, this finding was considered to be incidental.

In group 2, mean food consumption was similar to the control group.

1.6 FOOD CONSUMPTION - FEMALES
(See Figures on pp. 4-6, Summary Tables on pp. 35-37 and 40-42)

Pre-pairing, Gestation and Lactation Periods

Mean food consumption was similar in all groups and in all periods and no test item-related effects were observed. The overall mean food consumption per day e.g. in the pre-pairing period was 14.3 g, 13.5 g, 13.8 g and 13.8 g in order of ascending dose levels.

1.7 BODY WEIGHTS - MALES
(See Figures on pp. 7-9 and 14-16, Summary Tables on pp. 43-45, 51-53 and 59-61)

Pre-pairing, Pairing and After Pairing Periods

In group 4, mean body weight gain was slightly reduced after treatment start (+3.5% compared to +6.2% in the control group from day 1 - 8 of the pre-pairing period) and remained reduced until the end of the period. This reduction, which was on single days statistical significant, was considered to be test item-related but not adverse. Mean body weights were similar to the control group during the whole treatment period.

In group 3, mean body weight gain was slightly lower during the second week of the pre-pairing period (+2.5% compared to the control group from day 8 - 14 of the pre-pairing period). As a consequence, mean body weights were minor lower during the following periods. These effects were considered not to be test item-related but a result of biological variability.

In group 2, mean body weights and mean body weight gain were similar to the control group.

1.8 BODY WEIGHTS - FEMALES
(See Figures on pp. 10-13 and 17-20, Summary Tables on pp.46-50, 54-58 and 62-64)

Pre-pairing, Pairing, Gestation and Lactation Periods

Mean body weights and mean body weight gain were similar in all groups and in all periods and no test item related effects were observed. The overall mean body weight gain e.g. in the pre-pairing period was +9.0%, +5.9%, +10.0% and +9.4% in order of ascending dose levels.


2 CLINICAL LABORATORY INVESTIGATIONS
2.1 HEMATOLOGY
(See Summary Tables on pp. 65-66; Historical Data on pp. 67-68)

No test item-related effects were noted for males and females. The occasionally statistically significantly altered values were either in the range of the historical control data or no dose dependent manner was noted.

2.2 CLINICAL BIOCHEMISTRY
(See Summary Tables on pp. 69-70; Historical Data on pp. 71-72)

No test item-related effects were noted for males and females. The occasionally statistically significantly altered values were either in the range of the historical control data or no dose dependent manner was noted.

Exceptionally in group 4 males, the level of triglycerides was statistically significantly increased and not covered by the attached historical control data (0.91 nmol/l versus 0.50 nmol/l in the control group). Since no other test item-related altered parameters were noted in these group and the common 95% tolerance limit for males in this age was between 0.20 - 1.14 nmol/l, this finding was considered to be incidental.


3 REPRODUCTION AND BREEDING DATA
3.1 MATING PERFORMANCE AND FERTILITY
All females were mated within the first pairing period.

The median and mean precoital times were not affected by treatment with the test item. Mean precoital times were 2.7, 3.1, 2.7 and 2.9 days in order of ascending dose levels. The median precoital time was 2, 4, 3 and 3 days in order of ascending dose level.

One female each in group 3 and 4 was incidentally not pregnant. Therefore, the fertility index was 90% in these groups and 100% in group 1 and 2.

3.2 DURATION OF GESTATION
The mean duration of gestation was not affected by treatment with the test item. Mean duration of gestation was 21.5, 21.3, 21.7 and 21.7 days in order of ascending dose level.

3.3 CORPORA LUTEA COUNT
Mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (13.0, 13.9, 14.8 and 14.8 in order of ascending dose level) and gave no indication of a test item-related effect.

3.4 IMPLANTATION RATE AND POST-IMPLANTATION LOSS
The mean number of implantations per dam and the mean implantation loss was similar in all groups and not affected by treatment with the test item. The mean implantation loss per dam was 0.6, 0.4, 1.6 and 0.9 in order of ascending dose levels.

3.5 LITTER SIZE AT FIRST LITTER CHECK
The mean number of living pups at first litter check was similar in all groups (11.2, 12.8, 12.6 and 13.0 pups in order of ascending dose levels). No dead pups were found at first litter check in any group.

3.6 POSTNATAL LOSS DAYS 0 - 4 POST PARTUM
The mean postnatal loss was not affected by treatment with the test item. In group 3, one male pup was incidentally found dead on day 2 post partum. As a result, the mean postnatal loss between days 0 - 4 post partum was 0 in group 1, 2 and 4 and 0.1 in group 3.


4 TERMINAL FINDINGS - PARENTAL ANIMALS
4.1 ORGAN WEIGHTS
(See Summary Tables on pp. 73-78; Historical Data on pp. 79-102)

In males and females, no test item-related findings were noted on organ weights or organ weight ratios in any group.

In group 4 females, the body weight ratio of the liver was statistically significantly higher (3.33% compared to 3.00% in the control group). Since this value was in the range of the historical control data (3.11 - 3.63%) and no histopathological correlate was noted, this finding was considered to be incidental.

4.2 MACROSCOPICAL FINDINGS
(See Summary Tables on pp. 103-104)

In males and females, no test item-related macroscopical findings were noted in any group.

Due to the isolated occurrence and in the absence of a histopathological correlate following findings were considered to be incidental:

Group 1: Male nos. 4 and 5 had a dark red discolored thymus and female no. 44 had a left uterus horn reduced in size.

Group 2: Male no. 11 had yellowish discoloration on the mucosa of the pylorus region in the stomach and the right kidney was adherent to the liver and showed a pelvic dilation, male no. 12 had dark red discolorations on the meninges of the brain, male no. 13 had an isolated foci on the thymus, male no. 16 showed a left testis reduced in size and female no. 54 had reddish discoloration on the mandibular lymph node.

Group 3: Male no. 25 had yellowish discoloration on the mucosa of the pylorus region in the stomach.

Group 4: Male no. 33 had an urinary bladder with a milky-cloudy fluid content, male no. 35 had yellowish discoloration on the mucosa of the pylorus region in the stomach and male no. 36 showed seminal vesicles reduced in size.

4.3 HISTOPATHOLOGY FINDINGS
The test item LCE08129 produced no histological evidence of toxicological properties in the organs and tissues examined.

During sperm staging no differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: see "Remark"

Remarks:

In males at 1000 mg/kg body weight/day, mean food consumption and mean body weight gain were slightly reduced after treatment start. Mean body weights were similar to the control group. This transient reduction was considered to be test item-related but not adverse. At the dose levels of 300 and 1000 mg/kg bw/day, all animals pushed their head through the bedding material and showed salivation after application during the whole treatment period. These findings were considered to be a sign of discomfort but not a toxic effect. For further effects that were observed in males or in females at any dose level. NOAEL = highest dose tested.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item-related effects were noted in reproduction and development up to and including 1000 mg/kg body weight/day. NOEL = highest dose tested

Remarks on result:

other: Generation: reproduction and development (migrated information)

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

1 LITTER DATA - F1 PUPS
1.1 EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION
No abnormal findings were noted at first litter check or during the first 4 days post partum.

In group 3, each for one male pup and one female pup from the same litter a bloody wound on nose or cervical region was noted.

1.2 SEX RATIOS
Sex ratios at first litter check and on day 4 post partum were not affected by treatment with the test item. There was a statistical significant higher proportion of males in group 3 which was considered to be incidental due to the lack of dose-dependency.

1.3 BODY WEIGHTS TO DAY 4 POST PARTUM
Mean pup weights on day 1 post partum were not affected by treatment with the test item. On day 1 post partum mean pup weights were 6.3, 6.0, 6.2 and 6.4 g in order of ascending dose level.

Mean pup weight development until day 4 post partum was slightly lower in group 2 and 4 (+49.2%, +41.7%, +48.4% and +39.1% in order of ascending dose levels), but due to the lack of a dose-dependency and no statistical significant differences, this finding was considered to be a result of biological variability.

1.4 MACROSCOPICAL FINDINGS
No abnormal findings were noted at macroscopic examination of the pups in any group.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item-related effects were noted in reproduction and development up to and including 1000 mg/kg body weight/day. NOEL = highest dose tested

Remarks on result:

other: No test item-related effects were noted in reproduction and development up to and including 1000 mg/kg body weight/day. NOEL = highest dose tested

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item-related effects were noted in reproduction and development up to and including 1000 mg/kg body weight/day. NOEL = highest dose tested

Remarks on result:

other: No test item-related effects were noted in reproduction and development up to and including 1000 mg/kg body weight/day. NOEL = highest dose tested

Key result

Reproductive effects observed:

not specified

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

not specified

Dose response relationship:

yes

Relevant for humans:

yes

SUMMARY OF PERFORMANCE - P ANIMALS BREEDING FOR F1 LITTERS

Group

(mg/kg/day)

1

(0)

2

(100)

3

(300)

4

(1000)

Female numbers

41-50

51-60

61-70

71-80

Number of females paired

10

10

10

10

Number of females mated

10

10

10

10

Number of females,which were not pregnant (A)

0

0

1

1

Number of females which reared their pups until day 4 post partum

10

10

9

9

(A)  Female nos. 67 and 73 were not pregnant

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item LCE08129 to rats. LCE08129 was administered in olive oil as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. LCE08129 was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

Based on the results of this study a general NOAEL (No Observed Adverse Effect Level) was established at 1000 mg/kg body weight/day. The NOEL (No Observed Effect Level) for reproduction/ developmental toxicity was considered to be 1000 mg/kg body weight/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of LCE08129 on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

LCE08129 was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

The following dose levels were applied:

 

Group 1:              0 mg/kg body weight/day (control group)

Group 2:          100 mg/kg body weight/day

Group 3:          300 mg/kg body weight/day

Group 4:        1000 mg/kg body weight/day

 

A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (olive oil).

 

The following results were obtained:

 

 

PARENTAL ANIMALS

Mortality and General Tolerability

All animals survived until the scheduled necropsy.

 

In group 3 and 4, all animals pushed their head through the bedding material and showed salivation after application during the whole treatment period. These findings were considered to be a sign of discomfort but not a toxic effect.

 

Food Consumption and Body Weights

In group 4 males, mean food consumption and mean body weight gain were slightly reduced after treatment start. Mean body weights were similar to the control group. This transient reduction was considered to be test item-related but not adverse.

 

In group 2 and 3 males, mean food consumption, mean body weight gain and mean body weights were not affected by treatment with the test item.

 

In females, mean food consumption, mean body weight gain and mean body weights were not affected by treatment with the test item.

 

Functional Observational Batteryand Locomotor Activity

None of the parameters under investigation during the functional observational battery (appearance, behavior in the open field, grip strength, landing food splay, body temperature) and the locomotor activity measurements gave an indication of a test item-related effect.

 

Clinical Laboratory Investigations

No test item-related effects were noted from the clinical laboratory investigations.

 

Reproduction and Breeding Data

All pairs mated. No effects were noted in the reproduction and breeding data.

 

Organ Weights

No test item-related findings were noted on organ weights or organ weight ratios in any group.

 

Macroscopical Findings and Histopathological Examinations

No test item-related macroscopical or histopathological findings were observed.

 

 

LITTER DATA - F1 PUPS

Findings at First Litter Check and during Lactation

The mean number of pups at first litter check was not affected by the treatment with the test item. No abnormal pup was noted at any dose level during external examinations.

 

Pup Weights to Day 4 Post Partum

Pup weights were not affected by treatment with the test item.

 

Macroscopical Findings

No abnormal findings were noted during macroscopical examination of the pups.

 

Reference 3

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

without detailed documentation

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S5(R2) Detection of toxicity to reproduction for medicinal products and toxicity to male fertility

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

(no postnatal observations of pups)

GLP compliance:

not specified

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: (P) males 6-7 wks, females 10-11 weeks
- Weight at study initiation: (P) males 193-240 g; females 208-262 g
- Fasting period before study: no
- Housing: according to the investigators "during the acclimation and premating periods, 10 rats (5 males and 5 females) were housed per TR18 stainless-steel cage..."; during mating, 1 male and 1 female housed in RB3-modified high-grade polypropylene cage with stainless-steel mesh lids and floors; during gestation, 5 females per RB3-modified cage; after mating, 5 males per TR18 cage
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): expanded rodent diet (Special Diet Services Ltd.), ad libitum
- Water (e.g. ad libitum): public supply, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18
- Humidity (%): 55
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

other: 1% w/w aqueous Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- test material weighed into glass container and heated to ~80 deg C until molten
- vehicle heated to 75 deg C
- test material and vehicle combined using continuous magnetic stirring, 20% behenyl alcohol
- suspension cooled slowly to <60 deg C
- further cooled to 30 deg C
- slowly homogenized <=2 min
- cooled to room temperature
- 20% suspension prepared weekly
- 20% suspension provided top dose
- mid and low dose prepared on day of use by dilution with vehicle; 20% suspension magnetically stirred prior to removal of aliquots for dilution; dilutions hand swirled prior to magnetic stirring

VEHICLE
- Justification for use and choice of vehicle (if other than water): not stated
- Concentration in vehicle: 20, 2 and 0.2%
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): no data
- Purity: 1% aqueous

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: not stated
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of gestation
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: no data
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): 5/cage; RB3-modified cages
- Any other deviations from standard protocol: OECD guideline 415 recommends that: pregnant females are house individually, the mating period should be 3 weeks

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Males: from 71 days prior to mating, during mating and until females sacrificed
Females: from 15 days prior to mating, during mating, and up to day 17 of gestation; killed on day 20 of gestation

Frequency of treatment:

daily

Details on study schedule:

1-generation study

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: previous repeated dose toxicity study NOAEL was 1000 mg/kg bw/day
- Rationale for animal assignment (if not random): no data

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: evidence of reaction to treatment, moribund condition, mortality

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: males and females twice weekly prior to mating; males twice weekly after mating; females on gestation days 0, 3, 7, 10, 14, 18 and 20

FOOD CONSUMPTION :
- Food consumption: Yes
- Time schedule: males weekly prior to mating, females daily prior to mating, females on gestation days 0-2, 3-6, 7-9, 10-13, 14-17, 18-19

WATER CONSUMPTION: Yes
- Time schedule: males weekly prior to mating, females daily prior to mating, females on gestation days 0-2, 3-6, 7-9, 10-13, 14-17, 18-19

Oestrous cyclicity (parental animals):

10 days prior to mating, daily vaginal smears to assess regularity and duration of oestrus cycles

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight, sperm count in epididymides, sperm motility

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no, F1 generation examined as foetuses on day 20 of gestation

PARAMETERS EXAMINED
The following parameters were examined in parental females and F1 offspring: numbers of implantation sites, early and late resorptions and viable foetuses; distribution of foetuses in each uterine horn; placental weight; macroscopic examination of placentae; number and sex of foetuses

EXAMINATION OF PUPS: yes, for external and internal abnormalities (visceral and skeletal)

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following necropsy of the females
- Maternal animals: All surviving animals on day 20 of gestation

GROSS NECROPSY
- Gross necropsy of females consisted of reproductive endpoints only
- Gross necropsy of males consisited of macroscopic examination externally and internally; sperm assessment

HISTOPATHOLOGY / ORGAN WEIGHTS
No tissues were prepared for microscopic examination
Reproductive organs were weighed

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were examined on day 20 of gestation
- These animals were subjected to examination as follows: each foetus weighed; detailed external examination; contents of cervical, thoracic and abdominal cavities removed from half the foetuses and examined and sex recorded; these foetuses stained for skeletal examination; remaining foetuses examined for visceral abnormalities

HISTOPATHOLOGY / ORGAN WEIGTHS
No tissues prepared for microscopic examination or weighed.

Statistics:

One-way analysis of variance, t-tests - body weight, body weight change, food and water consumption; Dunnetts' or Behren's-Fisher's tests - organ weights; nested analysis of variance, weighted t-tests - foetal and placental weights

Reproductive indices:

number of pregnant females, fertility

Offspring viability indices:

number of viable young (offspring evaluated as foetuses on day 20 of gestation)

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

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- females, no mortality
- males, one death in top dose group in week 6, not considered to be treatment related in the absence of toxic signs in any other animals

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- no effects

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- no effects

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- PREGNANCY RATE - no effects (22, 22, 22 and 21 in control, low, mid and high dose groups respectively)
- no effects

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- no effects

ORGAN WEIGHTS (PARENTAL ANIMALS)
- males, reproductive organs, no effects

GROSS PATHOLOGY (PARENTAL ANIMALS)
- no effects

HISTOPATHOLOGY (PARENTAL ANIMALS)
- not examined

OTHER
- REPRODUCTIVE PARAMETERS
- Number of corpora lutea - no effects (17.8, 18.4, 18.7 and 18.9 for controls, low, mid and high dose respectively)
- Number of implantations - no effects (means 17.2, 17.0, 18.1 and 18.0 for controls, low, mid and high dose respectively)
- Number of viable young - no effects (means 16.4, 15.9, 17.0 and 16.9 for controls, low, mid and high dose  respectively)
- Sex ratio - no effects
- Number of resorptions (early or late) - no effects
- Pre-implantation loss - no effects (3.3, 8.3, 3.2,  5.8% for controls, low, mid and high dose respectively)
- Post-implantation loss - no effects (4.7, 6.4, 6.3 and 5.8% for controls, low, mid and high dose respectively)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Clinical signs:

not examined

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

VIABILITY (OFFSPRING)
- no effects

CLINICAL SIGNS (OFFSPRING)
- not applicable, foetuses examined at day 20 of gestation

BODY WEIGHT (OFFSPRING)
- no effects

SEXUAL MATURATION (OFFSPRING)
- not applicable, foetuses examined at day 20 of gestation

ORGAN WEIGHTS (OFFSPRING)
- not applicable, foetuses examined at day 20 of gestation

GROSS PATHOLOGY (OFFSPRING)
- not applicable, foetuses examined at day 20 of gestation

HISTOPATHOLOGY (OFFSPRING)
- not applicable, foetuses examined at day 20 of gestation

OTHER FINDINGS (OFFSPRING)
- MACROSCOPIC EXAMINATION (OFFSPRING)
- no variations were observed that were not comparable to historical control values
- SKELETAL EXAMINATION (OFFSPRING)
- no variations were observed that were not comparable to historical control values
- VISCERAL EXAMINATION (OFFSPRING)
- no variations were observed that were not comparable to historical control values

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Reproductive effects observed:

no

Conclusions:

In a reliable study, conducted to a protocol similar to OECD guideline 415, an NOAEL of 1000 mg/kg bw/day was determined in the rat for reproductive effects. The study was performed in compliance with GLP.

Executive summary:

[In view of the structural and chemical similarities, it is considered that the results of this study can be used for read-across to C12, 13, 14 and 15 alcohols; linear and monobranched.]

Reference 4

Endpoint:

two-generation reproductive toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Principles of method if other than guideline:

Weight of evidence based on several reproductive toxicity studies performed with read-across substances (C22 alcohol, APG C10-16) and based on a repeated dose toxicity study (OECD 408 + some endpoints of the OECD 416) performed with the registered substance

GLP compliance:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: not specified

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: not specified

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

1 000 mg/kg bw/day

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: not specified

Reproductive effects observed:

not specified

Conclusions:

So based on the following set of data, there is no need to perform a 2 generation reproductive toxicity study on the registered substance:
- Reproductive toxicity studies on the category:
OECD test 408 with parts of 416 on the registered substance,
2 screening for reproductive / developmental toxicity studies (OECD 422) performed with two members of the category (acetalization product between glucose and C14 alcohol and acetalization product between glucose and C20/22 alcohol)
- Toxicity profile and especially the conclusions on the potential reproductive toxicity of the two categories: C6-22 aliphatic alcohols and Alkyl polyglucosides
- 3 reproductive toxicity studies with source substances:
ICH S5(R2) guideline study and 26 week toxicity studies with C22 alcohol,
OECD guideline 421 with Alkyl polyglucosides, C12–C14 fatty alcohol

Executive summary:

Experimental data available on the registered substance, read-across substances and source substances:

• A pre-natal developmental study (OECD 414) with acetalization products between glucose and C16/18 (even numbered) -alcohol
• A combined repeated dose-reproduction study (OECD 422) with acetalization products between glucose and C14 alcohol
• A combined repeated dose-reproduction study (OECD 422) with acetalization products between glucose and C20/22 (even numbered) –alcohol
• A repeated dose 90-Day oral toxicity in Rodents (OECD Guideline 408) with parts of OECD 416, “Two-Generation Reproduction Toxicity Study” (only for clinical pathology and histopathological evaluations of male reproductive organs including sperm parameters) with acetalization products between glucose and C16/18 (even numbered) –alcohol.
• 3 reproductive toxicity studies with source substances:

ICH S5(R2) guideline study and 26 week toxicity studies with C22 alcohol

OECD guideline 421 with Alkyl polyglucosides, C12–C14 fatty alcohol

In all studies a NOAEL of 1000 mg/kg bw was derived for reproduction and developmental effects.

 Therefore the endpoints on fertility and reproduction are covered in a weight of evidence approach and there is no need to perform a 2 generation reproductive toxicity study.

Reference 5

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well-conducted study which meets basic scientific principles. The study is a read across from 1-docosanol (CAS 661-19-8).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline required

Principles of method if other than guideline:

Repeated dose toxicity test in which rats were orally dosed daily for 26 weeks and reproductive organs assessed

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: ~21-28 days at purchase
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: 5/cage in stainless steel cages, containing absorbent paper
- Diet (e.g. ad libitum): expanded rodent diet (Special Diets Services, UK), ad libitum
- Water (e.g. ad libitum): public supply (Suffolk Water Company, UK), ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 55
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

other: 1% aqueous Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- test material heated to approx. 80 deg C
- vehicle heated to approx. 75 deg C
- vehicle added to test material while being magnetically stirred at high speed
- resulting 20% (w/w) suspension homogenized and slowly cooled to below 60 deg C
- when cooled to 30 deg C, suspension slowly homogenized again for >=2 min
- cooled to room temp.
- stored at 13 deg C
- prepared once weekly
- 20% suspension used for top dose; for low and mid doses, suspension magnetically stirred and aliquots taken for dilution on day of use; constant dose volume of 5 ml/kg bw per dose
- dilutions mixed by hand swirling followed by magnetic stirring

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 1%
- Amount of vehicle (if gavage): 5 ml/kg bw per dose
- Lot/batch no. (if required): no data
- Purity: no data

Details on mating procedure:

No mating - screening test

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

26 weeks

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

No mating - screening test

Remarks:

Doses / Concentrations:
10, 100, 1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): random
- Other:
- Repeated dose toxicity (oral) study - acceptable as reproductive screen since reproductive organs were included in those evaluated

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS (including mortality): Yes
- Time schedule: >=twice daily
- Cage side observations included: evidence of reaction to treatment or moribund condition, evidence of ill health such as blood or loose faeces

DETAILED CLINICAL OBSERVATIONS: Yes, individual observations
- Time schedule: once daily during week 1, twice weekly during weeks 2 to 4, once weekly during weeks 5 to 13, once every 2 weeks from week 14 onwards

BODY WEIGHT: Yes
- Time schedule for examinations: pre-study, weekly during the study or more frequently if appropriate (for animals in moribund condition), at necropsy

FOOD CONSUMPTION:
- Food consumption for each cage determined: Yes

FOOD EFFICIENCY:
- Weekly group mean food conversion efficiencies calculated from the consumption and body weight gain data: Yes, for the first 14 weeks of treatment

WATER CONSUMPTION: No

OTHER: Ophthalmoscopic examination, haematology, clinical chemistry, urinalysis - reported elsewhere

Oestrous cyclicity (parental animals):

not examined; ovaries and uterus (with cervix) weighed and examined

Sperm parameters (parental animals):

not examined; testes and epididymides weighed and examined

Litter observations:

no litters - not mated - screening test

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: after 26 weeks of treatment
- Maternal animals: after 26 weeks of treatment

GROSS NECROPSY
- Yes

ORGAN WEIGHTS: adrenals, brain, kidneys, liver, lungs (with main stem bronchi), ovaries, pituitary, prostate, spleen, testes, thymus, thyroid (with parathyroids), uterus, cervix

HISTOPATHOLOGY: Yes - adrenals, brain, eyes, optic nerve, femur, heart, kidneys, liver, lungs, seminal vesicles, spinal cord, stomach, thyroid, uterus

Postmortem examinations (offspring):

no offspring - not mated - screening test

Statistics:

Bartlett's test for homogeneity of variance (organ weights, body weight changes); if significant, Behrens-Fisher test, otherwise Dunnett's test.
Two-tailed Fisher's exact test (macroscopic/microscopic pathological findings).

Reproductive indices:

not mated - screening test

Offspring viability indices:

no offspring - not mated - screening test

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

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

CLINICAL SIGNS AND MORTALITY
- one male in the mid-dose group died at week 25 (examination suggested aspiration of test material through mis-dosing; not considered to be treatment-related)
- no other clinical signs of systemic toxicity or mortality

BODY WEIGHT AND WEIGHT GAIN
- no effects

FOOD CONSUMPTION
- presumably no effects

FOOD EFFICIENCY
- no effects

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- not examined - screening test

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- not examined - screening test

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- not examined - screening test

ORGAN WEIGHTS (PARENTAL ANIMALS), including testis, epididymis, ovaries, uterus (with cervix)
- no effects

GROSS PATHOLOGY (PARENTAL ANIMALS), including testis, epididymis, ovaries, uterus (with cervix)
- no effects

HISTOPATHOLOGY (PARENTAL ANIMALS), including testis, epididymis, ovaries, uterus (with cervix)
- no effects

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: overall effects

Remarks on result:

other: Generation: not mated - screening study

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

no offspring - not mated - screening test

Reproductive effects observed:

not specified

Conclusions:

In a reliable screening study, a repeated oral dose NOAEL of 1000 mg/kg bw/day was determined for effects on reproductive organs in the rat. The result is a read across from 1-docosanol (CAS 661-19-8).

Reference 6

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to Guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

According to Guideline.

Route of administration:

oral: gavage

Vehicle:

not specified

Details on mating procedure:

- M/F ratio per cage: 1

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

2 weeks before pairing and continuously thereafter, up to the day before sacrifice (study day 53, day 4 post partum).

Frequency of treatment:

Daily

Remarks:

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

No. of animals per sex per dose:

10

Control animals:

yes

Positive control:

Not required.

Parental animals: Observations and examinations:

According to Guideline.

Oestrous cyclicity (parental animals):

According to Guideline.

Sperm parameters (parental animals):

According to Guideline.

Litter observations:

According to Guideline.

Postmortem examinations (parental animals):

According to Guideline.

Postmortem examinations (offspring):

According to Guideline.

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

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: No treatment-related effects.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Reproductive effects observed:

not specified

Conclusions:

Under the conditions of a state-of-the-art reproduction/developmental toxicity screening testing according to the OECD guideline 421, no adverse effects were observed regarding male and female reproductive organs even at the very high dose of 1000 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

A developemental toxicity study (OECD 414, GLP study) performed with the registered substance did not show any effect on the development of rats at the highest dose tested of 1000 mg/kg.

In a reliable study (Iglesais 2002) performed with the source substance docosan-1-ol and conducted according to a protocol similar to OECD guideline 414, a NOAEL for maternal toxicity, teratogenicity and foetotoxicity was determined in rabbits at 2000 mg/kg/day (highest dose tested). Another developmental toxicity study is available with the source substance behenyl alcohol (C22 alcohol):

1000 mg/kg/day is the NOAEL for maternal toxicity, teratogenicity and foetotoxicity in rats receiving behenyl alcohol by gavage for 15 days premating, during mating and up until gestation day 17. This is based on the absence of adverse effects in any of the parental, reproductive or foetal parameters examined. Based on these data, there is no need to perform additional test to evaluate the developmental toxicity on rats and rabbits of the registered substance.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 November 2007 - 25 August 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Except for absence of chemical analysis of the dosage forms

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch No. T70615
- Purity test date: 7 February 2009

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored at room temperature protected from sunlight.

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder
- Age at first treatment: 10-11 weeks
- Weight at first treatment (mean): 280 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Relative humidity: 50 ± 20%
- Light/dark cycle: 12h/12h
- Ventilation: 12 cycles/hour of filtered, non-recycled air.

IN-LIFE DATES: beginning: 3 December 2007 / end: 28 December 2007

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was ground, heated to 80°C, mixed with vehicle heated to 80°C, forming a solution.
The test item dosage forms were prepared daily

VEHICLE
- Justification for use and choice of vehicle (if other than water): lipophilicity of the substance.
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/day
- Lot/batch no. (if required): 057K6093
- Purity: not indicated

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Absence of a practical method of analysis

Details on mating procedure:

- Impregnation procedure: purchased time pregnant
- Proof of pregnancy: vaginal plug

Duration of treatment / exposure:

day 6 to day 20 post-coitum

Frequency of treatment:

once daily

Duration of test:

21 days

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day (f)
Basis:
other: nominal per gavage

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no effects on dams and development at up to 1000 mg/kg/day in a range-finding study, see 7.8.2
- Rationale for animal assignment: computerized stratification procedure (average body weight of each group is similar)

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule: regularly at 3- to 4-day intervals

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined daily: Yes

WATER CONSUMPTION: No

POST-MORTEM MACROSCOPIC EXAMINATION: Yes
- Sacrifice on gestation day# 21
- Examined: principal thoracic and abdominal organs

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of uterine scars, evaluation of placenta

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Other : number dead and live, body weight, sex

Indices:

% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites

Historical control data:

Not provided; not required for interpretation of the data obtained

Details on maternal toxic effects:

There were no treatement-related maternal toxic effects observed.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Not required (no effects)


There were no treatement-related embryotic ou teratogenic effects observed.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental toxicity

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

visceral/soft tissue: cardiovascular

Description (incidence and severity):

Several fetal variations were observed at soft tissue examination (absent innominate artery) and skeletal examination (ossification point on the 14th thoracic vertebra) but the incidences were low with no clear dose-relationship and there were no cartilage abnormalities. Variations at fetal examination are considered not to represent an adverse effect of development.

Key result

Developmental effects observed:

no

Not required (no effects)

Conclusions:

Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day for maternal and developmental toxicity.

Executive summary:

The test item, LCE07051, was administered daily, from day 6 to day 20 post-coitum, by the oral route (gavage), to mated female Sprague-Dawley rats at dose-levels of 100, 300 or 1000 mg/kg/day.

There were no test item-related mortalities nor any effects on maternal body weight gain or food consumption. Clinical signs were limited to salivation and several animals with soiled urogenital areas and were considered as non-adverse.

There were no treatment-related effects on pregnancy parameters (numbers of corpora lutea, implantation and live fetuses) nor on fetal body weight or sex.

There were no treatment-related fetal malformations. Several fetal variations were observed at soft tissue examination (absent innominate artery) and skeletal examination (ossification point on the 14th thoracic vertebra) but the incidences were low with no clear dose-relationship and there were no cartilage abnormalities. Variations at fetal examination are considered not to represent an adverse effect of development.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

without detailed documentation

Qualifier:

according to guideline

Guideline:

other: ICH Harmonized Tripartite Guideline S5 (R2) for Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

(non standard examination of soft tissue and head of foetuses)

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Froxfield SPF Rabbits Ltd., UK
- Age at study initiation: 18-26 weeks on arrival
- Weight at study initiation: 3.29-4.98 kg at start of study
- Fasting period before study: no data
- Housing: individually in suspended stainless-steel cages (TR6)
- Diet (e.g. ad libitum): standard rabbit diet (Special Diets Services Ltd., UK), ad libitum
- Water (e.g. ad libitum): public supply, ad libitum
- Acclimation period: >=1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18
- Humidity (%): 55
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

other: 1% Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- test material weighed into glass container and heated to ~80 deg C until molten
- vehicle heated to 75 deg C
- test material and vehicle combined using coninuous magnetic stirring, 20% behenyl alcohol
- suspension cooled slowly to <60 deg C
- further cooled to 30 deg C
- slowly homogenized <=2 min
- cooled to room temperature
- 20% suspension prepared weekly
- 20% suspension provided top dose
- mid and low dose prepared on day of use by dilution with vehicle; 20% suspension magnetically stirred prior to removal of aliquots for dilution; dilutions hand swirled prior to magnetic stirring

VEHICLE
- Justification for use and choice of vehicle (if other than water): not stated
- Concentration in vehicle: 20, 2 and 0.2%
- Amount of vehicle (if gavage): 10 ml/kg bw for vehicle control and top dose groups; 0.625 and 2.5 ml/kg bw for low and mid dose groups respectively
- Lot/batch no. (if required): no data
- Purity: 1%

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused with males of establised fertility
- If cohoused:
- M/F ratio per cage: no data
- Length of cohabitation: no data
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: no data
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: not specified, but referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no data

Duration of treatment / exposure:

days 6-19 of gestation

Frequency of treatment:

daily

Duration of test:

females killed on day 29 of gestation

Dose / conc.:

125 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female
Duration of test: 28 days
- Dose selection rationale: based on previous range-finding study
- Rationale for animal assignment (if not random): randomly allocated to the four treatment groups in order of mating "to evenly distribute the mated females among the groups"
- Other:
- approximately 2 weeks prior to arrival of females at testing facility, oestrus synchronised by supplier by intravenous injection of 25 IU luteinizing hormone
- following insemination, females injected intravenously with 25 IU luteinizing hormone to ensure successful ovulation
- examined on day 6 of gestation, prior to dosing, to determine suitability for use in study

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: evidence of reaction to treatment or moribund condition

DETAILED CLINICAL OBSERVATIONS: no data

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION: Yes
- Time schedule for examinations: days 1-5, days 6-12, days 13-19, days 20-23, days 24-28

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

POST-MORTEM EXAMINATIONS: yes, macroscopic examination
- Sacrifice on gestation day 29
- Organs examined in addition to uterine contents and ovaries: no data

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
- Number of viable young: males, females and total
- Distribution of foetusus in each uterine horn
- Uterus of any female presumed non-pregnant stained and examined for implantation sites

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter - cervical, thoracic and abdominal cavities dissected and contents examined microscopically
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: one third per litter
- Other: all per litter
- foetal body weight
- position of foetus in uterus
- placental weight

Statistics:

One-way analysis of variance, t-tests - body weight, body weight change, food and water consumption; Dunnett's or Behren's-Fisher's tests - organ weights; nested analysis of variance, weighted t-tests - foetal and placental weights

Indices:

no data

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
no effects other than pale faeces in animals of the top dose group

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
no effects

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

no

Table 1: Reproductive and developmental parameters

Observation	Dose (mg/kg bw/day)
	0	125	500	2000
Animals Assigned (Mated)	22	22	22	22
Animals Pregnant Pregnancy Rate (%)^a	20 91%	19 86%	19 86%	20 91%
Nonpregnant	2	3	3	2
Total litter loss (%)^a	1 10.0%	1 5.3%	1 5.3%	0 0.0%
Corpora Lutea/Dam (mean±SD)	12.8±3.1	12.9±2.2	12.6±3.0	12.2±3.9
Implantations/Dam (mean±SD)	11.4±3.9	11.1±2.6	11.0±3.3	10.6±4.3
Live Fetuses/Dam (mean±SD) Male (mean±SD) Female (mean±SD)	10.1±3.7 4.6±2.6 5.5±2.4	9.8±2.1 4.8±1.5 4.9±1.7	9.3^b±2.6 3.8±1.5 5.5±2.1	9.0±3.8 4.7±2.2 4.3±2.5
Resorptions/Dam (mean±SD) Early (mean±SD) Late (mean±SD)	1.4±1.2 0.4±0.6 1.0±1.0	1.3±1.2 0.3±0.5 1.1±1.0	1.7±1.3 0.4±0.6 1.2±1.1	1.6±1.2 0.7±0.8 0.9±0.9
Preimplantation Loss (%)	10.4	14.2	13.9	13.5
Postimplantation Loss (%)	12.1	12.0	15.2	14.7

^aCalculated for this table

^bIncludes one foetus not sexed at necropsy

Conclusions:

In a reliable study, conducted according to a protocol similar to OECD guideline 414, the NOAEL for maternal toxicity, teratogenicity and foetotoxicity in rabbits, was 2000 mg/kg/day (highest dose tested). The study was performed in compliance with GLP.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

without detailed documentation

Principles of method if other than guideline:

Method: other

GLP compliance:

not specified

Species:

rat

Strain:

Sprague-Dawley

Route of administration:

oral: gavage

Duration of treatment / exposure:

For 15 days prior to mating, during mating and up to Day 17 of gestation.

Frequency of treatment:

daily

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

Control animals:

yes

Details on study design:

Sex: female
Duration of test: 20th day of gestation

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

no

All female rats survived to sacrifice and no maternal toxicity was  observed. The were no differences between treated and control animals in  any of the rerpoductive endpoints investigated (corpora lutea, pre & post  implantation sites, early & late resorption sites).  The litter size,  foetal weight and sex ratio observed in treated groups was comparable to  the control group.  There were no unusual macroscopic findings among  foetuses. Microscopic examination did not show any increased incidence of  anomalies in skeletal or soft tissues. See above chapter 5.8.1 for  further details.

Conclusions:

1000 mg/kg/day is the NOAEL for maternal toxicity, teratogenicity and foetotoxicity in rats receiving behenyl alcohol by gavage for 15 days premating, during mating and up until gestation day 17. This is based on the absence of adverse effects in any of the parental, reproductive or foetal parameters examined.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Quality of whole database:

All the developemental toxicity studies are reliable

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Description of key information

Not available

Justification for classification or non-classification

Based on the toxicity to reproduction (OECD 422) and developmental toxicity studies (OECD 414, GLP, oral, rat) available with the registered substance and members of the category and supported by robust toxicity studies available on source substances, the registered substance is not classified according to the GHS.