ethyl N2-dodecanoyl-.sc.l.sc.-argininate hydrochloride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The influence of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) on reproductive performance when administered continuously in the diet through two successive generations of CD rats was performed meeting the requirements of the Organisation for Economic Co-operation and Development (OECD) Test 416 Guideline.

Key study: Test method equivalent to OECD 421 GLP study. A preliminary study of effects on reproductive performance in CD rats by dietary administration. It was concluded that a dietary concentration of 15000 ppm could be used as the highest treatment level for the two-generation study in the CD rat, equivalent to 1270/1330 mg/kg bw/day for males and females.

Key study: Test method according to OECD 416. GLP study. A two generation reproductive performance study by dietary administration to CD rats was conducted. It was concluded that the No Adverse Effect Level (NOAEL) for reproductive performance in the CD rat is 15000 ppm LAE equivalent to at least 1073 mg/kg bw/day based on the lowest average intake by adult rats before pairing and up to 2600 mg/kg bw/day for females during lactation. There was a slight reduction in offspring bodyweight gain just before weaning, a delay in vaginal opening of F1 females and reduced spleen weights among F1 and F2 offspring at 15000 ppm at a point when estimated achieved dosage would be in excess of 1900 mg/kg bw/day. However, these effects were transient and were regarded as not toxicologically significant.

Supporting study: A review regulator comments on the previous report of two generation reproductive performance, concludes that if the effects seen with LAE in the current study and in the preliminary study reflect an interaction with the prolactin axis. Then the significance of the observed delay in vaginal opening in the rat is reduced with respect to prospective risk to man.

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 11, 2003 to April 6, 2005

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 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: (P) approximately 38 to 42 days
- Weight at study initiation: (P) 138.2 to 250.2 g for males and 126.7 to 181.1 g for females
- Housing: Inside a barriered rodent facility designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms. Before each study the room was cleaned and disinfected with a bactericide. Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum of 12 days acclimatisation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-70%
- Air changes (per hr): 15 room air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light: 12-hour dark cycle

IN-LIFE DATES: From: March 5, 2003 To: April 29, 2005

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): The LAE was prepared for administration as a series of graded concentrations in the diet. The required amount of LAE was weighed into a suitable container and stirred together with an approximately equal amount of basal diet. This doubling process with basal diet was repeated until a total mixture of approximately 2 kg had been achieved. Portions of this premix were then added to appropriate weights of the basal diet (to the final weight required) and then mixed for a minimum of 6 minutes at 16 rpm in a Turbula mixer. The test substance was used as supplied.
- Mixing appropriate amounts with (Type of food): fortnightly
- Storage temperature of food: as required

Details on mating procedure:

- M/F ratio per cage: one-to-one basis with males from the same treatment group
- Length of cohabitation: maximum 3 weeks
- Proof of pregnancy: sperm in vaginal smear day 0 of pregnancy
- After successful mating each pregnant female was caged: Once mating had occurred, the males and females were separated and vaginal smearing discontinued.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations were analysed to assess the homogeneity and stability of the test material in the diet matrix. Samples of each formulation prepared for administration in Weeks 1, 11, 19, 30 and 35 of the study were analysed for achieved concentration of the test substance. The method of analysis was an adaptation of a method supplied by the Sponsor.

Duration of treatment / exposure:

Parental animals: 17-20 weeks
F1 animals: 19 weeks

Frequency of treatment:

Daily to each animal based upon the animal's bodyweight on that day

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 25 days of age.
- Age at mating of the mated animals in the study: 11-13 weeks

Dose / conc.:

0 ppm (nominal)

Dose / conc.:

2 500 ppm (nominal)

Dose / conc.:

6 000 ppm (nominal)

Dose / conc.:

15 000 ppm (nominal)

No. of animals per sex per dose:

Groups generation F0: 28 animals per sex per dose
Groups generation F1: 24 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary concentrations used in this study (0, 2500, 6000 and 15000 ppm) were selected with reference to previous work with this compound. In that study effects on pup survival and a delay in vaginal opening were observed at the highest treatment level, but effects were not so marked as to preclude the selection of 15000 ppm as the highest treatment level for the main multigeneration study in the rat.
- Rationale for animal assignment (if not random): The rat was chosen because it satisfies the requirement for a rodent species by regulatory agencies. The CD strain was used because of the background control data available in these laboratories.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes. In addition, observations relating to individual animals made throughout the day were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were inspected at least twice daily throughout the study and any visible signs of reaction to treatment were recorded, with details of type, severity, time of onset and duration. Approximately once each week all parental, and selected F1 animals were subjected to a thorough physical examination. In addition, a more detailed weekly physical examination to monitor general health was performed once each week for F0 animals and for F1 animals selected for continuation of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: F0 males were weighed on the day that treatment commenced (Week 0), at weekly intervals throughout treatment, and before necropsy. F0 females were weighed on the day that treatment commenced (Week 0), at weekly intervals until mating was detected, on Days 0, 6, 13 and 20 after mating, on Days 1, 4, 7, 14 and 21 of lactation and before necropsy (Day 28 post partum). F1 animals were weighed at the same frequency as F0 animals following selection at approximately 4 weeks of age.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. For the F0 generation, the weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for each week of treatment before the animals were paired for mating. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage. For each F0 female, the weight of food supplied, that remaining and an estimate of any spilled was recorded for the periods Days 0-5, 6-12 and 13-19 after mating and Days 1-3, 4-6, 7-13 and 14-20 of lactation. From these records the mean daily consumption (g/rat/day) was calculated for each animal. Food consumption for the F1 animals was recorded at the same frequency as F0 animals following selection, at approximately 4 weeks of age.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. The group mean achieved dosage for each sex, expressed as mg/kg bw/day, was calculated for each phase from the nominal dietary test material concentration, food consumption and bodyweight data.

OTHER:
From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were noted.

Oestrous cyclicity (parental animals):

For 15 days before pairing of the F0 and F1 generations, daily vaginal smears were taken from all females, using cotton swabs. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed. Daily vaginal smears taken from females, that reared their litters to weaning, before necropsy (Days 25 to 28 post partum) were used to determine the stage of the oestrous cycle at termination.

Sperm parameters (parental animals):

Immediately after scheduled sacrifice of each F0 and selected F1 male, the left vas deferens, epididymis and testis was removed and the epididymis and testis were weighed. The following tests were performed:
- Sperm motility: The percentages of motile and progressively motile sperm were reported for all groups.
- Sperm morphology: At least 200 sperm were assessed and the percentages of normal sperm and major categories of abnormal sperm were reported.
- Sperm count: Examination was limited to Groups 1 (Control) and 4 (15000 ppm) as no apparent affects were detected at the high dose level.
- Homogenisation-resistant spermatids count: Examination was limited to Groups 1 (Control) and 4 (15000 ppm) as no apparent effects were detected at the high dose level.

Litter observations:

PARAMETERS EXAMINED
All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter. Daily records were maintained for evidence of ill health or reaction to treatment; records were on an individual offspring basis or for the whole litter, as appropriate. Daily records were maintained of mortality and consequent changes in litter size from Days 1-21 and on Day 25 of age. On Day 4 of age, litters containing more than ten offspring were reduced to ten by random culling, leaving, whenever possible, five male and five female offspring in each litter. The sex ratio of each litter was recorded on Days 1, 4 (before and after culling) and on Day 21 of age. Individual offspring bodyweights were recorded on Days 1, 4 (before culling), 7, 14, 21 and 25 of age. Assessment of anogenital distance in the F2 offspring is a regulatory requirement where there has been evidence of an alteration in the timing of sexual maturation (in this case seen as a delay in the timing of vaginal opening).

GROSS EXAMINATION OF DEAD PUPS: Yes
All litters were examined in detail once each day from Day 1 to Day 21 of age for numbers of live and dead pups and also for general clinical signs and dam/litter interaction.

Postmortem examinations (parental animals):

SACRIFICE
F0 and selected F1 males were killed when the majority of litters had weaned [after at least 17 Weeks of treatment for the F0 males or at least 16 weeks of the F1 generation for the F1 males]. Females that littered and reared offspring to weaning were killed on Day 28 post-partum, after their respective litters had been weaned. Females that failed to mate were killed Day 25 after the last day of pairing. Females that failed to produce a viable litter were killed Day 25 after mating. Females whose litter died before Day 21 of lactation were killed on or after the day the last offspring died. Surplus F1 or F2 offspring were culled on Day 4 of age to leave 10 pups per litter. Other offspring, that were not selected to form the F1 generation, and F2 offspring were killed on Day 30 of age.

GROSS NECROPSY
All F0 and selected F1 adult animals were subject to a detailed necropsy, which involved the following: After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. For males, samples for sperm analysis were taken as soon as possible after death. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. The requisite organs were weighed and external and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative. For females, the numbers of implantation sites in each uterine horn was counted.

HISTOPATHOLOGY / ORGAN WEIGHTS
Microscopic examination was performed as follows: All tissues preserved for examination were examined for all F0/F1 adult animals of Groups 1 (Control) and 4 (15000 ppm) sacrificed on completion of the scheduled treatment period and for all animals killed or dying during the study. The reproductive organs (i.e. the right epididymis, prostate, seminal vesicles and right testis, or the cervix, ovaries, oviducts, uterus, and vagina) were examined from animals in Groups 2 (2500 ppm) and 3 (6000 ppm) that showed reduced fertility. This included males that failed to mate or failed to sire a pregnancy, and females that failed to mate, were not pregnant or failed to litter. Tissues reported at macroscopic examination as being grossly abnormal were examined for all F0/F1 adult animals.

The following organs taken from each F0 and F1 parental animal were weighed: adrenals, brain, epididymides, kidneys, liver, ovaries, pituitary, prostate-ventral, seminal vesicles (with coagulating gland, spleen, testes, thyroid with parathyroids, uterus with cervix and oviducts.

Postmortem examinations (offspring):

SACRIFICE
Offspring killed before Day 14 of age (including Day 4 culls) pentobarbitone. The sequence in which the animals were killed after completion of the scheduled period was selected to allow satisfactory inter-group comparison.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. For F1 and F2 offspring examined, a full macroscopic examination of the tissues was performed, after a review of the history. All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

HISTOPATHOLOGY / ORGAN WEIGTHS
For the assessment of the ovaries, one mid-line section of each ovary was examined for the presence of primordial follicles, growing follicles and corpora lutea. For F1 females, in addition to the general qualitative examination of ovarian tissue, a quantitative assessment was made of the primordial follicle population. For this, five sections were cut at about 100 µm intervals from the inner third of each ovary and the primordial follicles manually counted. For the assessment of the vagina, the stage of vaginal oestrus was evaluated based on vaginal epithelial morphology (and appearance of the uterus and endometrial glands). All other findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

The following organs taken from one male and one female F1 and F2 offspring per litter were weighed: brain, spleen, thymus.

Statistics:

Statistical analyses were performed to determine if apparent intergroup differences were statistically significant. For some parameters, the similarity of the data was such that analyses were not considered to be necessary. All statistical analyses were carried out separately for males and females. Data relating to food consumption was analysed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter parameters, the litter was considered to be the basic experimental unit.

The following data types were analysed at each timepoint separately: Bodyweight, gains over appropriate study periods; Mating performance and fertility; Sexual maturation, age and bodyweight at completion; Organ weights, both absolute and adjusted for terminal bodyweight; Startle response (F2 offspring).

For categorical data, including pathological findings, the proportion of animals affected was analysed using Fisher's Exact test (Fisher 1973) for each treated group versus the control.

For continuous data, Bartlett's test (Bartlett 1937) was first applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett's test, treated groups were then compared with the Control group, incorporating adjustment for multiple comparisons where necessary.

For organ weights, whenever Bartlett's test was found to be statistically significant, a Behrens-Fisher test was used to perform pairwise comparisons, otherwise a Dunnett's test was used.

The startle response data was analysed using a generalised mixed linear model with logit link function and the litter as a random effect (Lipsitz et al 1991). Each group was compared to Control using a Wald chi-square test.

Reproductive indices:

Mating performance and fertility: percentage mating (mating index), conception rate, fertility index, gestation index.

Offspring viability indices:

Survival indices: post-implantation survival index, live birth index, viability index, lactation index.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male in the 2500 ppm treatment group was killed for welfare reasons and one male was found dead in the 15000 ppm treatment group: histopathological examination revealed a malignant nephroblastoma. One female at 15000 ppm was killed for reasons of animal welfare after mating prior to implantation therefore pregnancy status was unconfirmed. These deaths were not considered to be treatment related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Overall bodyweight and bodyweight gain of F0 males were not adversely affected by treatment. Bodyweight gain for females before pairing was not affected by treatment. Bodyweight gain during gestation for the treated females were significantly greater than concurrent control values by between 11-19% but this finding is not considered to be adverse. The pattern of bodyweight gain during lactation was similar in all groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for male and female animals was similar for control and treated animals before pairing. Food consumption of females during gestation and lactation also showed no effects of treatment with LAE.

Food efficiency:

no effects observed

Description (incidence and severity):

Food conversion efficiency by males and females in the pre-pairing period was similar to the comparative Control animals.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no microscopic changes attributable to the administration of LAE. There were no obvious effects on sperm staging and further specific examinations were considered unnecessary.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Vaginal smears taken after weaning on Day 25-28 post-partum showed that treatment with LAE did not delay the return to a normal oestrous cycle, with all treated females showing oestrus before termination on Day 28 post-partum.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

A slight but non-significant reduction in the percentage of progressively motile sperm was observed in the 15000 ppm treatment group. No other effects on sperm parameters or homogenisation resistant spermatids were apparent.

Reproductive performance:

no effects observed

Description (incidence and severity):

Pre-coital interval and fertility were both unaffected by treatment. One female in each of the treated groups was not pregnant; this is within background expectation.
Gestation length and gestation index of F0 animals were unaffected by treatment at levels of up to 15000 ppm. The majority of females had gestation lengths within the range 22 to 23 days, one female treated with 2500 ppm had a longer gestation length but for the small litter size of four pups this in not unusual. There were no cases of dystocia.

Key result

Dose descriptor:

NOAEL

Effect level:

1 073 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Overall effects (before pairing)

Key result

Dose descriptor:

NOAEL

Effect level:

1 226 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Overall effects (Before pairing)

Key result

Dose descriptor:

NOAEL

Effect level:

1 518 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Overall effects (During gestation)

Key result

Dose descriptor:

NOAEL

Effect level:

2 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Overall effects (During lactation)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no signs observed that were considered to relate to treatment of F1 animals.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male at 15000 ppm was killed for humane reasons. From each of the 2500 and 15000 ppm treatment groups one female was killed for reasons of animal welfare after the offspring were weaned. These deaths were considered to be unrelated to treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Absolute bodyweights of males and females in the 15000 ppm group were marginally lower than Controls (3-5% lower) respectively, at the start of the generation, reflecting reduced bodyweight gain after Day 14 of age. Overall bodyweight change of males up to termination was unaffected by treatment. During the first week of the F1 generation bodyweight gains of females in the 15000 and 6000 ppm groups were significantly lower than Controls. All effects were only detectable at the points when achieved dosages were at their peak - over 1900 mg/kg bw/day for females receiving 15000 ppm in the diet.
During late gestation the bodyweights for females receiving 15000 ppm was marginally low when compared with Controls; this difference was made up during the latter half of lactation when bodyweight gain for females at 15000 and 6000 ppm was superior to that of Control animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Overall food consumption for animals before pairing and for females during gestation and lactation was unaffected by treatment with LAE.

Food efficiency:

no effects observed

Description (incidence and severity):

Overall food conversion efficiency for the period before pairing was unaffected by treatment with LAE.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weights (F1 adults)
There were no conclusive effects of treatment on organ weights. Absolute organ weights of after 16 weeks of treatment at the intermediate dosage of 6000 ppm males showed a reduced seminal vesicle weight (10%) this finding was considered incidental as the other dose groups were not affected and the relative weight was not affected. Females on Day 28 post-partum at 15000 ppm had reduced absolute relative spleen weight (9%), but relative weight was not significantly affected.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of F1 males after 16 weeks of treatment and F1 females on Day 28 post-partum did not reveal any observations that could be attributed to treatment

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no microscopic changes attributable to the administration of LAE. There were no obvious effects on sperm staging and further specific examinations were considered unnecessary.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no effects on F1 primordial ovarian follicle counts.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cycles – pre-mating
Oestrous cycles were unaffected by treatment.

Oestrous cycles – prior to termination
Vaginal smears taken after weaning on Day 25-28 post-partum showed that treatment with LAE did not delay the return to a normal oestrous cycle, with all treated females showing oestrus before termination on Day 28 post-partum.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

A slight but non-significant reduction in the percentage of progressively motile sperm was observed in the 15000 ppm treatment group. No other effects on sperm parameters or homogenisation resistant spermatids were apparent.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance and fertility, as assessed by the pre-coital interval and percentage mating, were unaffected by treatment with LAE at dietary concentrations up to 15000 ppm.
Gestation length and gestation index were unaffected by treatment with LAE at concentrations of up to 15000 ppm. Gestation lengths were within the range 22 to 23 days, with the exception of one female in the 15000 ppm group with a gestation length of 23.5 days – for the small litter size of 3 pups this is not unusual. There were no cases of dystocia.

Clinical signs:

no effects observed

Description (incidence and severity):

The general condition of the F1 offspring in the treated groups was similar to that of Control offspring.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The numbers of implantations, total litter size, live litter size and offspring survival, were generally similar in all groups and were considered to be unaffected by the level of LAE in the diet. At 15000 ppm one litter lost bodyweight between Days 1 and 4 of age and was terminated on Day 4 of age. At 2500 ppm one female also had a total litter loss and was observed to give birth to one dead pup, this isolated case was of no toxicological concern.

Body weight and weight changes:

not specified

Description (incidence and severity):

Offspring bodyweight
Bodyweight of the offspring at Day 1 was unaffected by the presence of LAE in the parental diet at concentrations of up to 15000 ppm. At 15000 ppm offspring bodyweight up to Day 14 was unaffected by treatment but a reduction in bodyweight gain from Day 14 was observed as the animals established independent feeding with animals being approximately 8% lighter than Controls by Day 25 of age.

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

Sexual maturation
Male sexual maturation, as assessed by age and bodyweight at completion of balano-preputial separation, was unaffected by treatment with LAE. Vaginal opening in animals receiving 15000 ppm LAE was significantly delayed by 4 days and bodyweight of these offspring at sexual maturation was significantly higher than for Control animals at a point when achieved dosage was calculated to be in excess of 1900 mg/kg bw/day. This finding was considered to be related to treatment but occurred at a time when achieved dosage at 15000 ppm was calculated to be in excess of 1900 mg/kg bw/day. There was no effect on the vaginal opening of females in the 6000 group, when achieved dosage was calculated to be in excess of 740 mg/kg bw/day, or in the 2500 ppm group.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weights (F1 weanlings)
At 15000 ppm absolute spleen weights for F1 male and female offspring were significantly lower than control (14 and 13% less respectively). For males, spleen weight relative to bodyweight was also significantly low. A reduction in absolute (10% lower) and bodyweight relative thymus weight of males treated at 15000 ppm and bodyweight relative thymus weight at 6000 ppm was also recorded. No other parameter attained statistical significance.

Gross pathological findings:

not specified

Description (incidence and severity):

Organ weights (F1 weanlings)
At 15000 ppm absolute spleen weights for F1 male and female offspring were significantly lower than control (14 and 13% less respectively). For males, spleen weight relative to bodyweight was also significantly low. A reduction in absolute (10% lower) and bodyweight relative thymus weight of males treated at 15000 ppm and bodyweight relative thymus weight at 6000 ppm was also recorded. No other parameter attained statistical significance.

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio, as assessed by the percentage of male offspring was unaffected by treatment and was close to the expected value of 50% in all groups.

Pre-weaning examinations
Surface righting, air righting and the auditory and visual responses of offspring were comparable in all groups and were not affected by LAE in the diet

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 356 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Overall effects (before pairing)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 489 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Overall effects (Before pairing)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 430 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Overall effects (During gestation)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 353 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Overall effects (During lactation)

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

General condition of offspring
The general condition of the offspring in the treated groups was similar to that of Control offspring.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Litter size and offspring survival
The numbers of implantations, total litter size, live litter size and offspring survival, were generally similar in all groups and were considered to be unaffected by the level of LAE in the diet. In the Control group one litter failed to thrive and was terminated on Day 2 of age. One litter in each of the 6000 and 2500 ppm groups were killed for reasons of animal welfare at or immediately following weaning as most of the offspring had died before Day 14 of age and surviving offspring were atypically small and considered unlikely to survive following the removal of the parent female.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Offspring bodyweight
Bodyweight of the offspring at Day 1 was unaffected by the presence of LAE in the parental diet at concentrations of up to 15000 ppm. At 15000 ppm offspring bodyweight up to Day 21 of lactation was unaffected by treatment. A small, but significant, reduction (7%) in cumulative bodyweight gain was recorded for the Day 1-21 period in both males and females, this had resolved by Day 25, with weight gains between Days 21 and 25 similar to Controls. Apparent effects on offspring bodyweight gain at 6000 ppm were largely attributable to a single litter and were considered to be of no toxicological importance.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Description (incidence and severity):

Offspring organ weights (F2 weanlings)
At 15000 ppm females attained a statistically significant reduction in absolute spleen weights (14%), when analysed relative to bodyweight the difference was not statistically significant. Males also showed a reduction in spleen weights of (12%) but this did not attain significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Offspring macropathology (F2 weanlings)
F2 offspring that died prior to scheduled termination did not show any changes that could be attributed to treatment. Macroscopic examination of the F2 offspring at scheduled termination showed no effects considered to be related to treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio and ano-genital distances
Sex ratio, as assessed by the percentage of male offspring, was unaffected by treatment and was close to the expected value of 50% in all groups.
Ano-genital distances showed clear distinctions between males and females, the distances recorded showed no differences between treated groups and Control animals.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Pre-weaning examinations
Group mean values for surface righting, air righting, startle response and the visual responses of offspring were not affected by maternal treatment. Poor performance in the startle response test in the F2 litters was principally due to a few litters with high incidences of failure at 6000 ppm (2/23) and 15000 ppm (2/24) and differences from Control were not statistically significant. Similar findings were not apparent in the first generation.

Developmental immunotoxicity:

not examined

Oestrus cycles – prior to termination
Vaginal smears taken after weaning on Day 25-28 post-partum showed that treatment with LAE did not delay the return to a normal oestrous cycle, with the majority of females showing oestrus before termination on Day 28 post-partum.

Key result

Reproductive effects observed:

no

Formulation chemistry:

The mean concentrations were between 6.4% above and 8% below nominal values, which were within applied limits +10%/-15%, confirming the accuracy of formulation.

Achieved dosages F0 generation:

Achieved intakes of LAE for F0 animals during the firts week of treatment were calculated as 1635/1687 mg/kg bw/day for males and females, respectively at the highest dietary concentration of 15000 ppm, with the lower treatment groups receiving intakes in proportion to the dietary concentrations. Test substance intake declined by about 52% for males and 44-45% for females by the time of pairing, consistent with the growth of the animals during the 10 week period. Intake remained relatively stable throughout gestation. Intake increased noticeably during lactation in response to the physiological demands of the litter, reaching a level of over twice the pre-pairing intake during the second week of lactation.

Achieved dosages F1 generation:

Achieved dosage for animals in the highest treatment group (15000 ppm) exceeded 2200 mg/kg bw/day during the first week after selection (age 4-5 weeks) and was similar to the F0 maternal values seen during the first week of lactation. Intake throughout the period

was higher than achieved for the F0 animals, reflecting the expected pattern of low bodyweight/high food consumption of the young animals.

Test substance intake declined by about 60-62 % for males and 51-54 % for females by the time of pairing consistent with the growth of the animals during the 10 week period. Intake remained relatively stable throughout gestation. Intake increased noticeably during lactation in response to the physiological demands of the litter.

 

Conclusions:

The objective of this study was to assess the influence of LAE on reproductive performance when administered continuously in the diet through two successive generations of CD rats. Results revealed that No Adverse Effect Level (NOAEL) for reproductive performance in the CD rat is 15000 ppm LAE equivalent to at least 1073 mg/kg bw/day.

Executive summary:

Conducted in accordance with the requirements of current, internationally recognised Good Laboratory Practice Standards, and following testing OECD Guideline 416, the objective of this study was to assess the influence of LAE on reproductive performance when administered continuously in the diet through two successive generations of CD rats.

 

The dietary concentrations used in this study (0, 2500, 6000 and 15000 ppm) were selected with reference to previous work with this compound. In that study effects on pup survival and a delay in vaginal opening were observed at the highest treatment level, but effects were not so marked as to preclude the selection of 15000 ppm as the highest treatment level for the main multigeneration study in the rat.

 

The influence of LAE on reproductive performance was assessed when administered continuously in the diet through two successive generations of Crl:CD® (SD) IGS BR rats. For the F0 generation, three groups of 28 male and 28 female rats received LAE orally, via the diet, at concentrations of 2500, 6000 or 15000 ppm for ten weeks before pairing, throughout pairing, gestation, lactation and until termination. A similarly constituted Control group received untreated basal diet for the same duration. The F1 generation comprised of 24 male and 24 female progeny from each group, and they continued to receive the relevant diet, as per the F0 generation, throughout the study until termination. The F1 generation was mated to produce the F2 generation which was raised to weaning and then the study was terminated.

 

During the study, data was recorded on clinical condition, bodyweight, food consumption, oestrous cycles, mating performance and fertility, gestation length and parturition observations. Seminology, organ weight, macroscopic and microscopic pathology investigations were undertaken on each adult generation. The clinical condition of offspring, litter size and survival, sex ratio, physical development, sexual maturation (selected F1 generation only) and bodyweight gain were assessed and organ weight and macroscopic pathology investigations were undertaken on each generation.

 

The general condition of F0 and F1 generation animals receiving diets containing LAE was similar to that of the Controls.

 

Bodyweight and bodyweight gain of adult F0 and F1 males and females were not adversely affected by treatment. At 15000 ppm bodyweight gains as F1 and F2 offspring began to eat the diet were slightly lower than Control offspring. Food consumption and food conversion efficiency was unaffected by the level of LAE in the diet in both generations.

 

There were no adverse effects in either generation on pre-mating oestrous cycles, mating performance, fertility, litter size, offspring survival and Day 1 bodyweight at levels of up to 15000 ppm of LAE in the diet. Pre-weaning examinations of surface, air righting startle response and pupil response for F1 and F2 offspring were not significantly affected by treatment.

 

Balano-preputial separation was unaffected at all dosage levels. A delay in vaginal opening of 4 days was recorded with treatment at 15000 ppm. The timing of vaginal opening occurs approximately within 2 weeks after the initiation of the selected F1generation. At this time, achieved dosages in the 15000 ppm group were 2269 and 1957 mg/kg bw/day. Treatment had no impact upon oestrous cycles pre-pairing or pre-termination, fertility or primordial follicle counts. The effect on vaginal opening appearsto be a transient effect at the time of highest exposure. The measurement of anogenital distance in the F2 offspring was also unaffected by treatment, confirming that LAE caused no changes in sexual differentiation.

 

Terminal investigations from F0 and F1adult animals showed no effects on pre-termination oestrous cycles or on sperm assessments. Macroscopic examination of adult animals and offspring revealed no changes attributable to treatment. In the 15000 ppm group absolute and/or bodyweight relative spleen weights of F0 and F1 females at scheduled termination and of F1 male and F1 female weanlings and F2 female weanlings on Day 30 of age were significantly lower than in controls. The magnitude of the difference reduced as age increased and was not accompanied by any macroscopic changes or microscopic changes in F0 and F1 adult animals so that the effect was therefore considered to be of no toxicological importance.

 

It is concluded that the No Adverse Effect Level (NOAEL) for reproductive performance in the CD rat is 15000 ppm LAE equivalent to at least 1073 mg/kg bw/day based on the lowest average intake by adult rats before pairing and up to 2600 mg/kg bw/day for females during lactation. There was a slight reduction in offspring bodyweight gain just before weaning, a delay in vaginal opening of F1 females and reduced spleen weights among F1 and F2 offspring at 15000 ppm at a point when estimated achieved dosage would be in excess of 1900 mg/kg bw/day. However, these effects were transient and were regarded as not toxicologically significant.

 

 

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 4, 2002 to July 3, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: (P) approximately 9-10 weeks of age
- Weight at study initiation: (P) 308 to 343g for males and 194 to 217g for females
- Housing: Rats were housed in TR18 cages from Arrowmight Biosciences, Hereford, England or RB3 modified and RB3 cages from North Kent Plastic Cages Limited, Rochester, Kent, England. The cages consisted of stainless steel (TR18) or high-density polypropylene (RB3 and RB3 modified) bodies with lids of stainless steel grid. TR18 and RB3 modified cages had stainless steel grid floors and were suspended in batteries over trays covered with absorbent paper which was replaced at least twice weekly or daily during pairing. RB3 cages had solid polypropylene floors and during the littering phase autoclaved wood shavings, which were renewed at least twice weekly, were provided as bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum of 5 days acclimatisation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23°C
- Humidity (%): 38-53%
- Air changes (per hr): 15 room air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light: 12-hour dark cycle

IN-LIFE DATES: From: October 14, 2002 To: January 29, 2003

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were usually prepared fortnightly, but more frequently as required. For each concentration the required amount of LAE was weighed into a suitable container. This was stirred together with an approximately equal amount of basal diet. This doubling process with basal diet was repeated until a total mixture of approximately 2 kg had been achieved. Portions of this premix were then added to appropriate weights of the basal diet (to the final weight required) and then mixed for a minimum of 6 minutes at 16 rpm in a Turbula mixer.

Details on mating procedure:

- M/F ratio per cage: one-to-one basis with males from the same treatment group
- Length of cohabitation: maximum 2 weeks
- Proof of pregnancy: sperm in vaginal smear day 0 of pregnancy
- After successful mating each pregnant female was caged: Once mating had occurred, the males and females were separated and vaginal smearing discontinued.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The suitability of the mixing procedure for LAE formulations and their stability were determined as part of this study. Homogeneity and stability of the LAE in UAR VRF1 Certified diet were assessed in a pre-treatment test mix at the anticipated lowest and highest concentrations to be used on the study (1000 and 20000 ppm). Samples taken from the top, middle and bottom layers of each concentration were analysed at time 0 (as at receipt at the analytical laboratory). Duplicate samples from each concentration were then analysed following 8 and 22 days storage at 21 °C.
Samples to assess achieved concentration at all dietary inclusion levels were taken from formulations prepared for the first week of treatment and from formulations prepared for feeding during the second week of treatment for the F1 generation. Duplicate samples were analysed.

Duration of treatment / exposure:

Parental animals: for 4 weeks prior to pairing, until termination after weaning of litters
F1 animals: 8 weeks

Frequency of treatment:

Daily to each animal based upon the animal's bodyweight on that day

Dose / conc.:

0 ppm (nominal)

Dose / conc.:

1 500 ppm (nominal)

Dose / conc.:

5 000 ppm (nominal)

Dose / conc.:

15 000 ppm (nominal)

No. of animals per sex per dose:

Groups generation F0: 8 animals per sex per dose
Groups generation F1: 12 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dietary concentrations were selected based on available toxicological data from a 13-week dietary study in Wistar Han ratsin which a concentration of 15000 ppm was associated with reduced bodyweight gain and 5000 ppm was identified as the No Observed Adverse Effect level (NOAEL).
- Rationale for animal assignment (if not random): The rat was chosen because it satisfies the requirement for a rodent species by regulatory agencies. The CD strain was used because of the background control data available in these laboratories.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were inspected at least twice daily throughout the study and any visible signs of reaction to treatment were recorded, with details of type, severity, time of onset and duration. Approximately once each week all parental, and selected F1 animals were subjected to a thorough physical examination.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed on the first day of treatment and from then twice weekly until termination. Females were weighed on the first day of treatment and then twice weekly until mating was detected. Subsequently the females were weighed on Days 0, 6, 13 and 20 after mating and on Days 1, 4, 7, 14 and 21 of lactation. Selected F1 animals were weighed twice weekly from a nominal 4 weeks of age until termination at approximately 8 weeks of age.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was recorded twice weekly for the F0 animals until they were paired for mating. Food consumption for the females was recorded for the periods 0-5, 6-12, 13-19 Days after mating and Days 1-3, 4-6, 7-13, 14-20 during lactation. Food consumption for the F1 selected animals was recorded twice weekly from nominal week 4 of age until termination.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes.
Group mean daily intakes and SD were calculated for females during gestation for the periods of Days 0-5, 6-12 and 13-19 and lactation for the periods of Days 1-3, 4-6, 7-13 and 14-20 including all females with live young at birth. It is considered that food consumed between Days 14 and 20 of lactation may include diet consumed directly by the offspring. For F0 pre-pairing animals and selected F1 animals, food consumption was calculated as a daily figure for all measurement intervals. During the maturation phases, when animals were gang housed, the derived mean food consumption and standard deviation are based on the assumption that each animal in the cage eats the same amount of diet. The quoted SD and n values represent the total numbers of animals and not the number of individual observations recorded.

Oestrous cyclicity (parental animals):

After pairing with the male vaginal wet smears were taken daily, by vaginal lavage, from all F0 females until evidence of mating was observed.

Litter observations:

PARAMETERS EXAMINED
All F1 offspring were examined at approximately 24 hours after birth (Day 1 of age) and the following were recorded for each litter: Number of offspring (live and dead); Individual offspring bodyweights; Sex ratio; Observations on individual offspring.
Daily records were maintained of mortality and consequent changes in litter size on Days 1-21 of age. The offspring were given individual within litter identification marks on Day 1 by toe tattoo. Litters were culled to 10 (5 males and 5 females when possible) on Day 4 of age. Offspring considered to be in poor condition/not eating were killed for humane reasons where appropriate.
The sex of the offspring was determined on Days 1 and 4 of age and at weaning.
The live offspring in each litter were weighed individually on Days 1, 4, 7, 14, 21 and 25 of age, and then twice weekly for selected F1 males and females.

GROSS EXAMINATION OF DEAD PUPS: Yes
All litters were examined in detail once each day from Day 1 to Day 21 of age for numbers of live and dead pups and also for general clinical signs and dam/litter interaction.

OTHER:
For each group the ratio of male to female offspring was calculated for all offspring at Day 1, and for live offspring on Days 1, 4 (before and after culling) and 21 of age. The ratio was expressed as the percentage of males from the total litter size.

Postmortem examinations (parental animals):

SACRIFICE
All animals were terminated at completion of the scheduled treatment periods. F0 animals males were killed after successfully littering by females. Females that littered and reared offspring to weaning were killed after their respective litters were weaned.

GROSS NECROPSY
- Gross necropsy consisted of: All animals were subjected to a detailed macroscopic examination for evidence of disease or adverse reaction to treatment. Samples of abnormal tissues were weighed (where considered to be of abnormal size) and retained in appropriate fixative. For the females, the number of implantation sites was also recorded.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed. Any early neonates that were found dead were, where possible, subjected to an external and internal macroscopic examination, with an assessment of the stomach for milk content. Any sporadic deaths in late neonates and F1 offspring which were not selected for the F1 generation were examined externally and internally for macroscopic abnormalities. Specimens of abnormal tissues were retained in retained in appropriate fixative.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. All F1 generation selected animals were subjected detailed macroscopic examination for evidence of disease or adverse reaction to treatment. Abnormal tissues were retained in retained in appropriate fixative.

Reproductive indices:

Mating performance and fertility: percentage mating, conception rate, fertility index, gestation length

Offspring viability indices:

Survival indices: post-implantation survival index, live birth index, viability index, lactation index.

Clinical signs:

no effects observed

Description (incidence and severity):

The general condition of animals receiving diets containing LAE was similar to that of the Controls throughout the generation.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No unscheduled deaths occurred.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Overall bodyweight and bodyweight gain of males were not adversely affected by treatment. Bodyweight gain for females before pairing was not clearly affected by treatment, although overall gain for the treated females was lower than control values there was no dosage dependent trend. Bodyweight gain during gestation and weight changes during lactation were similar in all groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for male and female animals was similar for control and treated animals before pairing. Food consumption of females during gestation and lactation also showed no effects of treatment with LAE. Food conversion efficiency by males and females in the prepairing period was similar to the comparative Control animals.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not specified

Description (incidence and severity):

Vaginal smearing was initiated at approximately 42 days of age for all groups, approximately 6 days after vaginal opening for controls, 5 days for low/intermediate treatment groups and 4 days after vaginal opening for the high level treatment group. The first recorded evidence of oestrus was seen at approximately 44 days of age in all groups, although earlier estrus may have occurred between the time of vaginal opening and the start of smearing. The first recorded oestrous cycle in the Controls was generally of 4 days duration, the normal mature cycle length in this strain, but treated animals showed a higher proportion of 5-day cycles for the first recorded cycle. However the subsequent cycle was reduced to 4 days in nearly all cases in all groups and it was considered that the delay in vaginal opening had no long lasting impact upon the normal sexual development of the female rats.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Pre-coital interval and fertility were both unaffected by treatment and all animals were fertile. Gestation length and gestation index were unaffected by treatment at levels of up to 15000 ppm: all females had gestation lengths within the range 22 to 23 days and there were no cases of dystocia.

Key result

Dose descriptor:

dose level: Highest treatment level for the main study (2G)

Effect level:

1 270 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: overall effects

Key result

Dose descriptor:

dose level: Highest treatment level for the main study (2G)

Effect level:

1 330 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: overall effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no significant clinical signs.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No deaths occured.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

BODY WEIGHT (OFFSPRING)
Bodyweight of selected F1 males and females at approximately four weeks of age and gains through to approximately eight weeks of age, were similar to the respective control group values and were considered to be unaffected by treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

SEXUAL MATURATION (OFFSPRING)
Balano-preputial separation was unaffected as assessed by age and bodyweight after treatment with LAE. Vaginal opening in animals receiving 15000 ppm LAE occurred approximately 4 days later than in the Control females. Bodyweight of these offspring at sexual maturation was also higher than Control animals, but there was no consistent treatment relationship between bodyweight and time of vaginal opening.
Vaginal smearing was initiated at approximately 42 days of age for all groups, approximately 6 days after vaginal opening for controls, 5 days for low/intermediate treatment groups and 4 days after vaginal opening for the high level treatment group. The first recorded evidence of oestrus was seen at approximately 44 days of age in all groups, although earlier estrus may have occurred between the time of vaginal opening and the start of smearing. The first recorded oestrous cycle in the Controls was generally of 4 days duration, the normal mature cycle length in this strain, but treated animals showed a higher proportion of 5-day cycles for the first recorded cycle. However the subsequent cycle was reduced to 4 days in nearly all cases in all groups and it was considered that the delay in vaginal opening had no long lasting impact upon the normal sexual development of the female rats.

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

GROSS PATHOLOGY (OFFSPRING)
Necropsy of the F1 offspring at approximately 8 weeks of age detected no macroscopic changes which were considered to be related to treatment.

Histopathological findings:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

VIABILITY (OFFSPRING)
The general condition of selected F1 animals from the treated groups was similar to that of the Controls.

Key result

Dose descriptor:

dose level: Highest treatment level for the main study (2G)

Generation:

F1

Effect level:

1 270 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: overall effects

Key result

Dose descriptor:

dose level: Highest treatment level for the main study (2G)

Generation:

F1

Effect level:

1 330 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: overall effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

F0: Achieved dosage

Achieved intakes of LAE during the first week of treatment were approximately 1270/1330 mg/kg/day for males and females respectively at the highest dietary concentration of 15000 ppm, with the lower treatment groups receiving intakes in proportion to the dietary concentrations. Test substance intake declined by about 25-26 % for males and 14-19% for females by the time of pairing consistent with the growth of the animals during the 4 week period. This decline continued during the second and third weeks of gestation. Intake increased noticeably during lactation in response to the physiological demands of the litter, reaching a level of over twice the prepairing intake during the second week of lactation.

F1: Achieved dosage

Achieved dosage for animals in the highest treatment group (15000 ppm) exceeded 2100 mg/kg/day during the first week after selection (age 4-5 weeks) and was similar to the F0 maternal values seen during the first week of lactation. Intake throughout the period was higher than achieved for the F0 animals, reflecting the expected pattern of low bodyweight/high food consumption of the young animals. The exposure level of F1 selected animals in the first two weeks after selection was approximately 50% higher than the average exposure for the F0 animals in the four weeks before pairing.

 

Conclusions:

This preliminary study describes the influence of Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE), when administered continuously in the diet, on reproductive performance in CD rats. The findings of this report were intended to enable selection of treatment levels for the two-generation study. For this purpose, LAE was administered orally via diet at concentrations of 1500, 5000 and 15000 ppm to groups of 8 males and 8 females for 4 weeks prior to pairing, until termination after weaning of the litters. Animals selected to form the F1 generation were continuously treated from about the time of weaning until terminated at approximately 8 weeks of age. It was concluded that a dietary concentration of 15000 ppm could be used as the highest treatment level for the two-generation study in the CD rat.

Executive summary:

This report describes a preliminary study performed to assess the influence of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE), when administered continuously in the diet, on reproductive performance in CD rats. The findings of this study were intended to enable selection of treatment levels for the two-generation study.

For this purpose, LAE was administered orally, via the diet, at concentrations of 1500, 5000 and 15000 ppm to groups of 8 males and 8 females for 4 weeks prior to pairing, until termination after weaning of the litters. Animals selected to form the F1 generation were continuously treated from about the time of weaning until terminated at approximately 8 weeks of age.

 

Inclusion of LAE in the diet at a level of 15000 ppm generated calculated intakes of the test material in the region of 1200 mg/kg/day for the parental animals during the four weeks of treatment before pairing and in excess of 1700 mg/kg bw/day for the selected F1 animals up to the age of 8 weeks.

 

After serial observations, the general condition of animals receiving diets containing LAE was similar to that of the Controls.

 

Bodyweight and bodyweight gain of F0 males and females were not adversely affected by treatment and there were no adverse effects on bodyweight gain for females during gestation and lactation. Offspring bodyweight at Day 1 of age, gain to weaning, and bodyweight of selected F1 males and females to eight weeks of age, were unaffected by treatment.

 

Food consumption was similar in all groups of F0 animals, both before mating and for females during gestation and lactation. Food consumption by selected F1 animals was similar to that of Controls. There were no consistent clear effects on food conversion efficiency in the females or males before pairing or in the F1 generation to 8 weeks of age.

 

At 15000 ppm achieved intakes of LAE at the start of F0 treatment were approximately 1270/1330 mg/kg bw/day and the overall average before pairing was 1150/1295 mg/kg bw/day for males/females respectively. The peak intake (approximately 3070 mg/kg bw/day) by females occurred during the second week of lactation. Selected F1 animals had higher achieved intakes than their F0 parents, averaging approximately 1750/1735 mg/kg bw/day for males/females respectively between ages of 4-8 weeks. Calculated intakes at lower treatment levels were in proportion to the dietary concentrations.

 

Mating performance, fertility, litter size, and growth were unaffected by the presence of LAE in the diet at levels of up to 15000 ppm. Sexual maturation in males was unaffected by treatment but vaginal opening was delayed by 4 days in females treated at 15000 ppm. Subsequent establishment of the normal oestrous cycles was demonstrated in all groups.

 

Necropsy of F0 parental animals, weanling offspring and selected offspring killed at approximately 8 weeks of age did not detect any effects of treatment.

 

There were no adverse effects on bodyweight, bodyweight change, food consumption and conversion efficiency at levels of up to and including 15000 ppm.

 

There were no apparent effects on mating performance, fertility and litter size at birth associated with the level of test material in the diet and the growth of the selected F1 offspring was satisfactory.

 

The effects of pup survival and delay in vaginal opening may be related to a reduction or inhibition of prolactin production at the highest treatment level, but effects were not so marked as to preclude the selection of 15000 ppm as the highest treatment level for the main multigeneration study in the rat.

 

It was concluded that a dietary concentration of 15000 ppm could be used as the highest treatment level for the two-generation study in the CD rat.

Reference 3

Endpoint:

toxicity to reproduction

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

June 12, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: An expert review.

Reason / purpose for cross-reference:

other: An expert review this study

Reason / purpose for cross-reference:

other: An expert review this study

Reproductive effects observed:

not specified

Conclusions:

Some evidence of poor peri-natal survival at the highest treatment level and it was suggested that this could be due to reduced milk production immediately after parturition and possibly relating to reduced prolactin levels. No adverse effect on litter survival was seen in the main study, but there was some reduction in offspring bodyweight gain which may relate to reduced prolactin and reduced milk output. If the effects seen with LAE in the current study and in the preliminary study reflect an interaction with the prolactin axis,then the significance of the observed delay in vaginal opening in the rat is reduced with respect to prospective risk to man.

Executive summary:

In the first draft of their review of the results of the two generation report on N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) the Food Standards Agency (London, England) and the National Food Institute (Seborg, Denmark) commented that "The effect on the vaginal opening of F1 females at 15000 mg/kg diet was related to treatment and consistent with the preliminary study, and therefore should not be disregarded. At 15000 mg/kg diet there was also an effect on the offspring body weight gain prior to weaning. Considering these effects together, the NOAEL was 6000 mg LAE/kg diet (equal to 434 mg LAE/kg bw/day). ".

 

The author would not disagree with the statements regarding the delay in vaginal opening being real and being treatment related at the highest dose level, but urge that consideration should be given as to whether this is an "adverse" effect and to the achieved dose levels at the time that this particular response was generated.

Within this study the only indication of an effect upon reproductive performance was on the timing of vaginal opening in animals receiving 15000 ppm LAE. Vaginal opening in the F1 females was significantly delayed (P<0.01) by 4 days. This was not related to general delays in growth as the bodyweight of these offspring at sexual maturation was higher (P<0.01) than for Control animals at a point when vaginal opening occurred. Throughout the study there were no detectable effects upon estrous cycles or fertility or upon the timing of sexual maturation in the F1 males. Measurement of the anogenital distance of the F2 offspring did not detect any evidence of change in either males or females which might suggest that LAE had any effect on sexual development.

 

The OECD guideline on Prenatal Developmental Toxicity (1998) includes an annex which describes an "adverse effect" as follows:

"any treatment-related alteration from baseline that diminishes an organism's ability to survive, reproduce or adapt to the environment."

 

It is considered that the effect of LAE on the timing of vaginal opening was a transitory effect which had no lasting effect upon sexual development or reproductive performance of the female rat. The effect upon vaginal opening should not, therefore, be considered an adverse response when it considering the parameters to set critical levels for the NOAEL values.

Slight reductions in offspring bodyweight gain before weaning were also unsupported by continued effects during maturation and may be attributed to the exceptionally high exposure levels achieved during the late lactation/early post weaning period.

 

The timing of vaginal opening in the rat depends on increasing output of estradiol from the ovaries under the control ofincreasing levels of follicle stimulating hormone (FSH) and luteinising hormone (LH). Exogenous agents which affect levels of these hormones can affect the timing of vaginal opening. Thus estrogens and the insecticide kepone, which has estrogenic effect,will advance the timing of vaginal opening in the rat. Conversely the phytoestrogen, genistein, and TCDD will delay the vaginal opening, presumably by interacting with the release of FSH and LH.

It is concluded that the delay in vaginal opening in the highest dose group was related to the exceptionally high exposure received by females at this stage of development. The effect was, however, transitory and there was no evidence of adverse effects upon any other reproductive parameters which would be expected if the delay in sexual maturation were tobe considered as an "adverse response". The NOAEL level can therefore be set as the highest level of exposure to LAE and should not be based on the effect upon the timing of vaginal opening.

In the rat there is also evidence that prolactin can affect the timing of vaginal opening - injection of prolactin advances the timing of vaginal opening and bromocriptine (which inhibits prolactin secretion) delays vaginal opening. These effects may be the result of prolactin affecting the levels of estrogen. Dopaminomimetic drugs, as a class, have also been found to reduce prolactin levels and can thus delay the timing of vaginal opening.  In the rat, prolactin has functions in lactation and in maintaining functional corpora lutea in early pregnancy but in man itsfunctions appear to be limited to mammary development and it is much less important to reproduction. If LAE is acting via an effect upon prolactin then it would probably have low significance to man.

In the preliminary study with LAE, there was some evidence of poor peri-natal survival at the highest treatment level and it was suggested that this could be due to reduced milk production immediately after parturition and possibly relating to reduced prolactin levels. No adverse effect on litter survival was seen in the main study, but there was some reduction in offspring bodyweight gain which may relate to reduced prolactin and reduced milk output. If the effects seen with LAE in the current study and in the preliminary study reflect an interaction with the prolactin axis, then the significance of the observed delay in vaginal opening in the rat is reduced with respect to prospective risk to man.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 073 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 1. This study was carried out in accordance with internationally valid GLP principles.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Results from reports assessed, revealed that No Adverse Effect Level (NOAEL) for reproductive performance in the CD rat is 15000 ppm LAE equivalent to at least 1073 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Rat

Tolerance study: Test method equivalent to OECD 414. GLP study. A study of tolerance in the rat by oral gavage administration was assessed to the test substance N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE). It was concluded from this investigation that the highest dosage for use in a preliminary embryo-fetal study in the rat should be 2000 mg/kg bw/day (the highest dosage that could be achieved for this species without exceeding without exceeding guideline figures for volume dosage).

Preliminary study: Test method equivalent to OECD 414. GLP study. In this preliminary study, the influence of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) was assessed in sexually mature rats of the CD strain, to establish suitable dosages for a main embryo-fetal toxicity study.It was concluded that 2000 mg/kg bw/day of LAE would be suitable as the highest dose level for a main embryo-fetal study in the rat. This dosage is considered to be the maximum achievable dosage in relation to formulation and volume dosage under the condition of use.

Key study: Test method equivalent to OECD 414. GLP study. Astudy of embryo-fetal toxicity in the CD rat by oral gavage administration was assessed to study the influence upon the progress and outcome of pregnancy in sexually mature rats of the CD strain. There were no apparent treatment related effects upon fetal survival, growth or development. Therefore, it was concluded, that 200 mg/kg/day was the no-adverse-effect-level (NOAEL) for the dam but 2000 mg/kg bw/day was the NOAEL for the fetuses of dams that survived to the end of pregnancy.

Rabbit:

Tolerance study: Test method equivalent to OECD 414. GLP study. A study of tolerance in the rabbit by oral gavage administration was performed to assess the effects of repeated oral administration of LAE to to non-pregnant and pregnant rabbits in order to establish the maximum suitable dosage for use in a preliminary study of embryo-fetal development. Treatment at 1000 mg/kg bw/day for 7 consecutive days did not result in any significant effect on embryo survival. This would ensure that the effects of LAE on the pregnant rabbit were investigated at a dosage which is commonly accepted as a maximum limit dosage in studies of this type.

Preliminary study: Test method equivalent to OECD 414. GLP study. A preliminary embryo-fetal toxicity study in the rabbit by oral gavage administration was assessed to establish suitable dosages for a main embryo-fetal toxicity study in the rabbit. On the basis of this preliminary study 250 mg/kg/day was considered to be the no-effect-level (NOEL) for the mother, but both 500 and 1000 mg/kg bw/day were associated with reduced food consumption and reduced bodyweight gain. 1000 mg/kg bw/day was considered to be the NOEL for the fetus. A dosage of up to 1000 mg/kg/day would be suitable as the highest dosage level for a main embryo-fetal study in the rabbit.

Key study: Test method equivalent to OECD 414. GLP study. A LAE study of embryo-fetal toxicity in the rabbit was performed to observe the test substance by oral gavage administration upon the progress and outcome of pregnancy was assessed in sexually mature rabbits of the New Zealand White strain.It was concluded that, despite the slightly higher risk of irritation to the respiratory tract at concentrations of 60 mg/ml and above (dosages of 300 and 1000 mg/kg bw/day), 300 mg/kg bw/day was the no-adverse-effect level (NOAEL) for the dam and 1000 mg/kg bw/day was the NOAEL for the fetus.

Supporting study:Interpretation of the data derived from the rat embryo-fetal study by the Study Director,revealed that on the basis of these observations, the author considered that the No-Adverse-Effect-Level (NOAEL) for the dam was 200 mg/kg bw/day, although the NOAEL for the fetus was 2000 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 23, 1998 to August 6, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

other: Preliminary study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: approximately 10 to 11 weeks of age
- Weight at study initiation: 217 to 267 g
- Housing: Rats were housed in TR18 cages from Arrowmight Biosciences, Hereford, England and RB3 modified cages from North Kent Plastic Cages Ltd, Erith, Kent, England. The cages consisted of stainless steel (TR18) or high density polypropylene (RB3) bodies with lids and floors of stainless steel grid and were suspended in batteries over trays covered with absorbent paper which was replaced twice weekly or daily during pairing.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum of 5 days acclimatisation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-70%
- Air changes (per hr): 15 room air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light: 12-hour dark cycle

IN-LIFE DATES: From: May 26, 1998 To: June 20, 1998

Route of administration:

oral: gavage

Vehicle:

other: Methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): freshly each day

VEHICLE
- Justification for use and choice of vehicle (if other than water): Methylcellulose
- Concentration in vehicle: suspension in 1% Methylcellulose

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Information on the homogeneity of mixing, stability and concentration of the test material in vehicle was determined. Specimen formulations of 600 ml were prepared at each of the highest and lowest concentrations. These were then separated into 3 x 200 ml aliquots for each concentration. Aliquot 1 was stored at 21 ºC and analysed at one, four and 48 hours; aliquots 2 and 3 were stored at 4 ºC and analysed at 48 hours and eight days respectively. Concentration of test material in formulations was analysed. On one day for each of the first and last weeks of dosing, 4 x 2ml samples were taken from each group. Two samples for each group were analysed for test material content.

Details on mating procedure:

Females were paired on a one-to-one basis with stock males of the same strain. Each morning following pairing, the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each female and examined for the presence of spermatozoa. The day on which a sperm positive vaginal smear or at least three copulation plugs were found was designated Day 0 of gestation.

Duration of treatment / exposure:

From Day 6 to 19 of gestation.

Frequency of treatment:

Daily to each animal based upon the animal's bodyweight on that day

Duration of test:

From May 26, 1998 to June 20, 1998

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

200 mg/kg bw/day

Dose / conc.:

600 mg/kg bw/day

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

22 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages of 200, 600 and 2000 mg/kg bw/day were based on results from a preliminary study where it was concluded that dosages of up to 2000 mg/kg bw/day had no significant effect upon maternal food consumption, bodyweight gain, embryo-fetal survival or development. The 14 day dosing period was chosen to encompass the period of organogenesis and fetal development.
- Rationale for animal assignment (if not random): Rat was chosen because it satisfies the requirement for a rodent species by regulatory agencies. The Charles River Crl: CD® BR rat strain was used because of the historical control data available in this laboratory.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice daily throughout the study and any visible signs of reaction to treatment were recorded, with details of type, severity, time of onset and duration.

BODY WEIGHT: Yes
- Time schedule for examinations: Females were weighed on Days 0, 3 and 6 to 20 inclusive after mating. Group mean values and SD were calculated on Days 0, 3 and 6 to 20 inclusive of gestation. Weight changes were plotted graphically with respect to Day 6 of gestation. In the absence of any effects upon maternal weight or fetal weight no adjustments were made to allow for the weight of the gravid uterus.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was recorded for the periods Days 0-2, 3-5, 6-8, 9-11, 12-15, 16-17 and 18-19 after mating.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. For each animal, mean daily intakes were calculated for periods of Days 0-2, 3-5, 6-8, 9-11, 12-15, 16-17 and 18-19 of gestation. Group mean daily intakes and SD were calculated for these periods.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Each animal was first examined macroscopically for evidence of disease or adverse reaction to treatment and specimens of abnormal tissues were retained.

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

Fetal examinations:

- External examinations: Yes. Each fetus was weighed, sexed and examined for any external abnormalities. Individual placental weights and placental abnormalities were recorded.
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
- Other: The neck and the thoracic and abdominal cavities of approximately half of each litter were dissected and examined. All fetal abnormalities were recorded and the offspring eviscerated prior to fixation in industrial methylated spirit.

Statistics:

Formal statistical analysis was not performed as there were no intergroup differences suggestive of treatment related effect.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The general condition of the surviving animals was satisfactory and all the females were pregnant.
Noisy respiration was seen during the treatment period in three animals receiving 200 mg/kg/day, and in a total of 7 animals at 600 and 9 animals at 2000mg/kg/day (including animals which were killed prematurely).
Salivation at the time of dosing was seen in all animals receiving 2000mg/kg/day on approximately 50% of dosing occasions reaching peak daily incidence at about Day 14 of gestation. Fourteen animals receiving 600mg/kg/day showed occasional incidences of salivation during the dosing period, and at 200mg/kg/day salivation was seen in only one animal on one occasion.
Neither noisy respiration nor salivation were seen in the Control group.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Three females receiving 2000mg/kg/day (F68, F77 and F87) were killed in extremis on Days 7 or 8 of gestation (the second or third day of treatment). All three animals showed signs of noisy and gasping respiration, and salivation after dosing. Two females (F77 and 87) showed bodyweight loss before termination and F87 showed signs of underactive behaviour and piloerection. Necropsy revealed large amounts of gaseous material in the stomach in F68 and F77 and in F87 the entire gastro-intestinal tract was distended with gas. In addition F68 had enlarged and prominent lymph nodes, and F77 had haemorrhagic lungs, large amounts of pale yellow viscous material in the ileum, reduced and dehydrated caecal contents, dark and enlarged adrenals and a pronounced internal structure of the kidneys. All animals were pregnant.
Two females at 600mg/kg/day (F54 and F59) were similarly affected towards the end of gestation, both showing signs of noisy respiration, salivation at the time of dosing and bodyweight losses. F54 was killed for humane reasons and F59 was killed in extremis, both had reached Day 17 of gestation. Necropsy of these animals revealed that gastro-intestinal tract was distended with gaseous material. Both animals had grossly normal implantations.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no overall treatment related effects upon bodyweight. Occasional animals in all groups receiving LAE showed transient bodyweight losses for periods following commencement of treatment at Day 6 and some animals receiving 600 mg/kg/day also showed weight losses towards the end of treatment. Many of the cases of weight loss coincided with episodes of respiratory distress. There were no similar bodyweight losses in the Control group

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no overall treatment related effects upon food consumption, group mean values were similar for all groups. However there were occasional animals in the treatment groups which showed periods of reduced food intake, which appealed to be associated with episodes of respiratory distress.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no maternal necropsy findings which were considered to be related to treatment.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no effects on fetal survival as indicated by the extent of pre- and post-implantation loss and the numbers of lives fetuses.

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no effects on fetal survival as indicated by the extent of pre- and post-implantation loss and the numbers of lives fetuses.

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

not specified

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Severe respiratory distress was recorded at a low frequency among animals receiving 600 or 2000 mg/kg bw/day. Three females had to be killed after 2 or 3 days of dosing at 2000 mg/kg bw/day and two females had to be killed after 11 or 12 doses at 600 mg/kg/day. Necropsy of these animals did not detect damage to the lungs and gross changes were limited to accumulation of gas within the gastro-intestinal tract. This may relate to gasping respiration following possible aspiration of increased secretions and/or traces of the dosing material following treatment with the more concentrated/viscous suspensions at the higher doses.

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

clinical signs

mortality

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: Clinical signs, mortality

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal and placental weights, and the incidences of fetal abnormalities and variants were unaffected by treatment.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified

Reduction in number of live offspring:

not specified

Changes in sex ratio:

not specified

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Description (incidence and severity):

Fetal and placental weights, and the incidences of fetal abnormalities and variants were unaffected by treatment.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Fetal and placental weights, and the incidences of fetal abnormalities and variants were unaffected by treatment.

Visceral malformations:

no effects observed

Description (incidence and severity):

Fetal and placental weights, and the incidences of fetal abnormalities and variants were unaffected by treatment.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

There were no obvious treatment related effects upon fetal development as assessed by fetal weight and macroscopic examination at necropsy.

Key result

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

The results of the analyses of the concentration of test material in formulations confirm acceptable homogeneity and stability and that concentrations of analysed preparations from the first and last weeks of treatment were satisfactory.

Conclusions:

Assessment of influence on progress and outcome of pregnancy in CD rats in order to establish suitable dosages for a main embryo-fetal toxicity study of Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) by oral gavage administration. It was concluded that 200 mg/kg bw/day was the no-adverse-effect-level (NOAEL) for the dam and 2000 mg/kg bw/day was the NOAEL for the fetus.

Executive summary:

The influence of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) upon the progress and outcome of pregnancy was assessed in sexually mature rats of the CD strain. For this purpose, LAE was administered by gavage at dosages of 200, 600 or 2000 mg/kg bw/day to groups of 22 presumed pregnant rats from Day 6 to 19 after mating, inclusive. Control animals received the vehicle, 1% w/v Methylcellulose, throughout the same period.

All surviving females were killed on Day 20 after mating for examination of their uterine contents followed by detailed fetal examination.

Severe respiratory distress was recorded at a low frequency among animals receiving 600 or 2000 mg/kg bw/day: three females had to be killed after 2 or 3 days of dosing at 2000 mg/kg/day and two females had to be killed after 11 or 12 doses at 600 mg/kg bw/day. Necropsy of theseanimals did not detect damage to the lungs and gross changes were limited to accumulation of gas within the gastro­intestinal tract. This may relate to gasping respiration following possible aspiration of increased secretions and/or traces of the dosing material following treatment with the more concentrated/viscous suspensions at the higher doses. Under the circumstances the respiratory distress seen in animals receiving 600 or 2000 mg/kg/day is considered not to be a systemic toxic response to oral ingestion of LAE. It is thought to be unlikely to extrapolate to man but may suggest possible bronchial irritation if the test material is inhaled.

 

With the exception of transient effects on bodyweight and food consumption associated with individual animals showing respiratory distress, at 600 and 2000 mg/kg bw/day, there were no adverse effects of treatment on the mother and there were no adverse effects on fetal survival and development at dosages of up to 2000 mg/kg bw/day.

There were no overall treatment related effects on bodyweight or food consumption, although occasional animals in treatment groups showed periods of bodyweight loss and reduced food intake which were related to-respiratory distress.

There were no apparent treatment related effects upon fetal survival, growth or development.

It was concluded, because of the maternal deaths at 600 and 2000 mg/kg bw/day, that 200 mg/kg bw/day was the no-adverse-effect-level (NOAEL) for the dam but 2000 mg/kg bw/day was the NOAEL for the fetuses of dams that survived to the end of pregnancy.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 20, 1998 to March 23, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

other: Preliminary study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: approximately 18 to 26 weeks old
- Weight at study initiation: 2.94 kg to 4.34 kg
- Housing: Rabbits were housed singly in suspended anodised aluminium cages (Type TR6) mounted in batteries (NKP Cages LTD, Erith, Kent, England) and were randomly distributed on the battery in order to equalise, as far as possible, environmental influences among the groups. The cages were fitted with perforated counter-sunk floor panels, and an undertray beneath the floor was lined with absorbent paper, which was changed at least three times per week.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days acclimatisation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-23°C
- Humidity (%): 40-70%
- Air changes (per hr): at least 12 air changes per hour
- Photoperiod (hrs dark / hrs light): a 14-hour light: 10-hour dark cycle

IN-LIFE DATES: From: August 2, 1998 To: September 3, 1998

Route of administration:

oral: gavage

Vehicle:

other: Methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
LAE was prepared as a suspension in 1% Methylcellulose and dosages were of the material as supplied. The volume of suspension was prepared sufficient for up to 5 days of use and the bulk was divided into daily aliquots which were stored at approximately 4 °C until use.

DIET PREPARATION
- Rate of preparation of diet (frequency): freshly each day

VEHICLE
- Concentration in vehicle: suspension in 1% Methylcellulose

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of test material in formulations used in this study was analysed on one day for each of the first and last weeks of dosing. Four 2 ml samples were taken from each group and two samples for each group were analysed for test material content. Analysis could be extended to the spare samples as a check on analytical results and any surplus samples were stored at approximately -20°C pending possible requirement for re-analysis before being discarded following satisfactory completion of the study.

Details on mating procedure:

Females were time-mated with New Zealand White males of established fertility at Charles River U.K. Ltd. The day of mating was designated Day 0 of gestation. Each female was identified by a unique animal number in the ear and records of parentage and the identity of the male partner were supplied.

Duration of treatment / exposure:

From Day 6 to 19 of gestation.

Frequency of treatment:

Daily to each animal based upon the animal's bodyweight on that day

Duration of test:

From August 2, 1998 to September 3, 1998

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

22 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages of 100, 300 and 1000 mg/kg bw/day were based on results from a preliminary embryo-fetal toxicity study where it was concluded that dosages of 500 and 1000 mg/kg/day had adverse effects upon the mother but no adverse effects were detected on embryo-fetal survival or development; 1000 mg/kg bw/day was also considered to be the maximum practical dosage in the rabbit, based on the limitations of the formulation and practicable dose volume.
- Rationale for animal assignment (if not random): The rabbit was chosen because it satisfies the requirement for a non rodent species by regulatory agencies. The New Zealand White strain was used because of the historical control data available in this laboratory.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice daily throughout the study and any visible signs of reaction to treatment were recorded, with details of type, severity, time of onset and duration.

BODY WEIGHT: Yes
- Time schedule for examinations:Females were weighed daily throughout the study and bodyweights are reported for Days 0, 6, 8, 10,12,14,16,18, 20, 24 and 28 of gestation. Bodyweights recorded on intermediate days are retained with the raw data.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was recorded for the periods 1-5 (2-5 for animals received from the supplier on Day 2 after mating), 6-12, 13-19, 20-23, and 24-28 inclusive after mating.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. For each animal, mean daily intakes were calculated for periods of Days 1-5 (2 -5 for animals received from the supplier on Day 2), 6-12, 13-19, 20-23 and 24-28 of gestation. Group mean daily intakes and SD were calculated for these periods.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: Each animal was first examined macroscopically for evidence of disease or adverse reaction to treatment and specimens of abnormal tissues were retained.

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

Fetal examinations:

- External examinations: Yes. Each fetus was weighed, sexed and examined for any external abnormalities. Individual placental weights and placental abnormalities were recorded.
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
- Other: The neck, thoracic and abdominal cavities of all fetuses from each litter were dissected and examined, and the sex of each fetus recorded and position within the litter recorded. Following examination the fetuses were eviscerated and one third of the fetuses in each litter were decapitated and the heads fixed in Bouins fluid. These heads were then subject to free-hand serial sectioning and the serial sections examined for abnormalities. Torsos and the remaining intact fetuses were fixed in industrial methylated spirits for subsequent skeletal examination.

Statistics:

The significance of intergroup differences was assessed using the Startox programme. Dependent on the heterogeneity of variance between treatment groups, parametric tests (analysis of variance, Snedecor and Cochran 1967) followed by Williams' test (Williams' 1971/2) or non-parametric tests, (Kruskal-Wallis, Hollander and Wolfe 1973) followed by Shirley's test (Shirley 1977) were used to analyse these data, as appropriate. For litter data the basic sample unit was generally the litter and, due to preponderance of non-normal distributions, non-parametric analyses were routinely used. All significant (i.e. p<0.05) inter-group differences from the control are reported only where supported by a significant analysis of variance (i.e. p<0.05).

Description (incidence and severity):

Ninety animals were initially allocated to study (including two animals in Group 4 which were replaced early in treatment because of difficulties with dosing and poor acclimatisation). Both of the replaced animals (F69R and F70R) showed evidence of congestion in the lungs and frothy liquid in the trachea. Eighty eight animals were allowed to continue with treatment and 76 of these were pregnant and carried a live litter to termination (Day 29 of gestation).

Reactions to dosing were largely limited to changes in respiration pattern seen in 5 animals at 300 mg/kg/day and 5 animals at 1000 mg/kg/day, including the two animals (F69R and F70R) which were killed early in the study and replaced. Adverse respiratory reactions were believed to be associated with a higher risk of irritation being induced during the dosing procedure when high concentrations of test material were used; difficulties with dosing were much reduced when the surface of the catheter was washed clean rather than wiped dry before dose administration. There were no other signs of adverse reaction to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

not specified

Description (incidence):

At 1000 mg/kg/day one animal (F71) was killed for reasons of animal welfare on Day 9 of gestation following periods of noisy respiration accompanied by reduced food consumption and faecal output, and an aqueous discharge in the cage undertray on Day 9 of gestation. Necropsy revealed a small amount of frothy liquid in the trachea, and congestion in the lungs. One animal (F60) at 300 mg/kg/day was killed for reasons of animal welfare on Day 14 of gestation, because of gasping respiration following dosing. Necropsy revealed incomplete collapse of the lungs, with occasional dark areas of change on the lung surfaces.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Bodyweight gain of animals receiving 1000 mg/kg/day was slightly but significantly lower than that of the Controls throughout most of the treatment period. This was considered to be treatment related, because the animals showed recovery of weight gain once treatment was completed, although interpretation was complicated by the fact that animals allocated to the high dosage group had gained more bodyweight than Controls in the period between mating and start of treatment.
Bodyweight gains of animals receiving 100 or 300 mg/kg/day was similar to that of the Controls throughout gestation.

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

Food consumption by animals receiving 1000 mg/kg/day fell slightly when treatment started and was significantly lower than that of the Control group during the period Days 13-19 of gestation but recovered to similar to Control levels after completion of the dosing period. Food consumption at 100 and 300 mg/kg/day was unaffected by treatment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no necropsy findings for females killed on Day 29 after mating which were considered to be related to treatment with LAE.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

There were no apparent treatment related effects on fetal survival. The numbers of corpora lutea， implantations and live young in the Control group were generally lower than in the treated groups but intergroup differences were not statistically significant.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no effects of treatment on fetal weight or placental weight. There was a low incidence of fetal anomalies seen at necropsy or at subsequent detailed examinations but no indication of any adverse effect of treatment.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
At 1000 mg/kg bw/day one animal (F71) was killed for reasons of animal welfare on Day 9 of gestation following periods of noisy respiration accompanied by reduced food consumption and faecal output, and an aqueous discharge in the cage undertray on Day 9 of gestation. Necropsy revealed a small amount of frothy liquid in the trachea, and congestion in the lungs. One animal (F60) at 300 mg/kg bw/day was killed for reasons of animal welfare on Day 14 of gestation, because of gasping respiration following dosing. Necropsy revealed incomplete collapse of the lungs, with occasional dark areas of change on the lung surfaces. Female no. 84 at 1000 mg/kg bw/day aborted on Day 24 of gestation: necropsy revealed three empty implantation sites in the left uterine horn, but no implantations in the right horn of the uterus. Two dead fetuses, both of which appeared grossly normal, were found in the undertray of the cage.

Reactions to dosing were largely limited to changes in respiration pattern seen in 5 animals at 300 mg/kg bw/day and 5 animals at 1000 mg/kg bw/day, including the two animals (F69R and F70R) which were killed early in.

The numbers of corpora lutea, implantations and live young in the Control group were generally lower than in the treated groups but intergroup differences were not statistically significant.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not specified

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no obvious treatment related effects upon fetal development as assessed by fetal weight and macroscopic examination at necropsy.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: fetotoxicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Assessment of influence on progress and outcome of pregnancy of N-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) by oral gavage administration was studied in sexually mature rabbits of the New Zealand White strain. All surviving females were killed on Day 29 after mating for examination of their uterine contents followed by detailed fetal examination. Results revealed that 300 mg/kg bw/day was the no-adverse-effect level (NOAEL) for the dam and 1000 mg/kg/day was the NOAEL for the fetus.

Executive summary:

The influence of N-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) by oral gavage administration upon the progress and outcome of pregnancy was assessed in sexually mature rabbits of the New Zealand White strain.

 

For this purpose, LAE was administered by gavage at dosages of 100, 300 or 1000 mg/kg bw/day to groups of 22 presumed pregnant rabbits from Day 6 to 19 after mating, inclusive. Twenty two females designatedas Control animals received the vehicle, 1% w/v Methylcellulose, throughout the same period.

 

All surviving females were killed on Day 29 after mating for examination of their uterine contents followed by detailed fetal examination.

 

Results have shown that signs of reaction to treatment were largely associated with dosing difficulty and respiratory signs in a few animals at 300 and at 1000 mg/kg bw/day. These signs were believed to be attributable to the physical nature of the highly concentrated suspensions and were largely alleviated by using a clean moist catheter (rather than a clean dry catheter) for dose administration. One animal at each of 300 and 1000 mg/kg bw/day were killed for humane reasons and necropsy revealed some lung congestion. A further animal at 1000 mg/kg/day aborted on Day 24 of gestation but the majority of animals were pregnant and between 17 and 22 animals had live litters for evaluation at Day 29 after mating.

 

Bodyweight gain of animals receiving 1000 mg/kg bw/day was slightly but significantly lower than that of the Controls during the treatment period. Food consumption was low during the second week of treatment. Both bodyweight gain and food consumption recovered to be similar to Control levels once treatment ceased. Bodyweight gain and food consumption were unaffected at 100 and 300 mg/kg bw/day.

 

Necropsy of the females and detailed fetal examinations at completion of pregnancy revealed no treatment related effects upon the dam or upon the litter and development of the fetus.

 

It was concluded that, despite the slightly higher risk of irritation to the respiratory tract at concentrations of 60 mg/ml and above (dosages of 300 and 1000 mg/kg bw/day), 300 mg/kg bw/day was the no-adverse-effect level (NOAEL) for the dam and 1000 mg/kg bw/day was the NOAEL for the fetus.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

December 19, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: An expert review.

Reason / purpose for cross-reference:

other: The record is an expert review of the cross-referenced studies.

Reason / purpose for cross-reference:

other: The record is an expert review of the cross-referenced studies.

Reason / purpose for cross-reference:

other: The record is an expert review of the cross-referenced studies.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Overview of three key studies (Study of embryo-foetal toxicity in the CD rat by oral gavage; Study of embryo-foetal toxicity in the rabbit by oral gavage; 13-week study) assessed by an expert: Proposal to base the NOAEL on results of dietary studies.

Executive summary:

Data derived from the rat embryo-fetal study revealed there were cases of increased salivation and associated respiratory distress after oral gavage dosing at both 2000 and 600 mg/kg bw/day. This resulted in the premature termination of three animals early in the treatment period at 2000 mg/kg bw/day and of two animals after an extended treatment period at 600 mg/kg bw/day. On the basis of these observations it is considered that the No-Adverse-Effect-Level (NOAEL) for the dam was 200 mg/kg bw/day, although the NOAEL for the fetus was 2000 mg/kg bw/day.

 

It was considered that the respiratory distress was not due to a systemic toxic response to oral ingestion of LAE and it was thought that the reaction was unlikely to be repeated in man although it may suggest possible bronchial irritation if the test material were inhaled.

 

In the rabbit embryo-fetal study there was also evidence that oral gavage administration of LAE at concentrations of 60 or 100 mg/ml could cause difficulties with dose administration and respiratory signs. These problems could largely be alleviated by cleaning and lubricating the dosing catheter, showing that the effect was related to the physical process of administration rather than systemic toxicity. On this basis 300 mg/kg bw/day was considered to be the NOAEL in the rabbit.

 

In a later 13-week rat study, LAE was administered by the dietary route at levels of up to 50000 ppm (equivalent to an average intake of 3714 mg/kg bw/day for males or 3915 mg/kg bw/day for females) for 13 weeks and there were no cases of lung damage or death. A dietary concentration of 5000 ppm (equivalent to an average intake of 384 mg/kg bw/day for males or 445 mg/kg/ bw/day for females) was identified as the NOAEL in this study.

 

As the respiratory effects following oral gavage were considered to be an artefact of the route of administration and not the result of systemic toxicity, and the fact that the dietary route is the more common method of administration for assessing the toxicity of this type of material it is considered more appropriate to use the data from dietary study in setting the definitive NOAEL. On the basis of studies performed this would mean that the appropriate NOAEL should be considered to be 384 mg/kg bw/day rather than 200 mg/kg bw/day for the rat or 300 mg/kg bw/day for the rabbit.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 24, 1998 to August 6, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: (P) approximately 10 to 11 and 13 to 14 weeks of age for Groups 1 and 2 respectively
- Weight at study initiation: (P) Females: 226 to 235 for Group 1 and 242 to 262 g for Group 2
- Housing: Rats were housed in TR18 cages from Arrowmight, Biosciences, Hereford, England and RB3 modified cages from North Kent Plastic Cages Limited, Erith, Kent, England. The cages consisted of stainless steel (TR18) or high density polypropylene (RB3) bodies with lids and floors of stainless steel grid and were suspended in batteries over trays covered with absorbent paper which was replaced twice weekly or daily during pairing.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum of 6 days acclimatisation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-25°C
- Humidity (%): 40-70%
- Air changes (per hr): 15 room air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light: 12-hour dark cycle

IN-LIFE DATES: From: February 18, 1998 To: March 26, 1998

Route of administration:

oral: gavage

Vehicle:

other: Methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): freshly each day

VEHICLE
- Justification for use and choice of vehicle (if other than water): Methylcellulose
- Concentration in vehicle: 1% w/v Methylcellulose

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- M/F ratio per cage: one-to-one basis with stock males of the same strain
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no

Duration of treatment / exposure:

Group 1:
Day 1-2: 250 mg/kg/day
Day 3-4: 500 mg/kg/day
Day 5-6: 1000 mg/kg/day
Day 7-8: 2000 mg/kg/day

Group 2:
From Day 6 to 12 of gestation: 2000 mg/kg/day

Frequency of treatment:

Daily to each animal based upon the animal's bodyweight on that day

Duration of test:

From February 18, 1998 to March 26, 1998

Dose / conc.:

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

Remarks:

Staircase phase (group 1)

Dose / conc.:

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

Remarks:

Staircase phase (group 1)

Dose / conc.:

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

Remarks:

Staircase phase (group 1)

Dose / conc.:

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

Remarks:

Staircase phase (group 1)

Dose / conc.:

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

Remarks:

Constant dosage phase (group 2)

No. of animals per sex per dose:

Staircase phase: 4 females.
Constant dosage phase: 4 females.

Control animals:

no

Details on study design:

STUDY SCHEDULE
1. Staircase phase in non-pregnant females: Four non-pregnant females were allocated to this phase of the investigation. Treatment commenced at an initial dosage of 250 mg/kg bw/day for two days. For subsequent two-day periods the dosage was doubled until reaching the maximum practical dosage of 2000 mg/kg bw/day (based on the physical properties of the formulated suspension).

Dosages received on the various days of the study were as follows:
Day of treatment 1-2 3-4 5-6 7-8
Dosage (mg/kg/day) 250 500 1000 2000

2. Constant dosage phase in pregnant females:
Following consultation with the Sponsor, four females were mated with proven males of the same stock and dosed on Days 6 to 12 of gestation inclusive at 2000 mg/kg bw/day. Animals were terminated on Day 13 of gestation.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes.
Each animal was examined macroscopically for evidence of disease or adverse reaction to treatment and specimens of tissues considered abnormal were retained in an appropriate fixative. Mated animals were checked for pregnancy.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed and examined daily throughout the study and any visible signs of reaction to treatment were recorded with details of type, severity, time of onset and duration.


POST-MORTEM EXAMINATIONS: Yes
- Organs examined: each animal was examined macroscopically for evidence of disease or adverse reaction to treatment and specimens of tissues considered abnormal were retained in an appropriate fixative.

Clinical signs:

no effects observed

Description (incidence and severity):

Staircase phase : Group 1 : non-pregnant females
The general condition of the females was unaffected by treatment. Salivation was recorded on number of occasions for a short period immediately after dosing. The frequency of recorded salivation was increased at dosages of 1000 and 2000 mg/kg/day.

Constant dosage phase: Group 2: pregnant females
The general condition of the females was unaffected by treatment and no deaths occurred. Salivation was recorded occasionally for all animals for a short period immediately after dosing toward the end of the treatment period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

Staircase phase : Group 1 : non-pregnant females
No deaths occured.

Constant dosage phase: Group 2: pregnant females
No deaths occured.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Staircase phase : Group 1 : non-pregnant females
Bodyweight and bodyweight gain in the non-pregnant female was essentially unaffected by treatment with LAE at dosages of up to 2000 mg/kg/day. At the highest dose (2000 mg//kg/day) three of the four animals showed minor losses in bodyweight after the first dose but weight gain was recovered the following day.

Constant dosage phase: Group 2: pregnant females
Bodyweight and cumulative bodyweight gain in the pregnant female appeared to be unaffected by treatment with LAE at a dosage of 2000 mg/kg/day.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Staircase phase : Group 1 : non-pregnant females
No adverse findings were recorded at necropsy of the non-pregnant females after a total of 8 doses of LAE which escalated from 250 to 2000 mg/kg/day.

Constant dosage phase: Group 2: pregnant females
All females were pregnant at termination and no adverse findings were recorded at necropsy of the pregnant females after a total of 7 doses of LAE at 2000 mg/kg/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

not examined

Pre- and post-implantation loss:

not examined

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

not examined

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

not examined

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
SACRIFICE
All animals were terminated at completion of the scheduled treatment periods.

GROSS NECROPSY
- Gross necropsy consisted of: . Each animal was examined macroscopically for evidence of disease or adverse reaction to treatment and specimens of tissues considered abnormal were retained in an appropriate fixative. Mated animals were checked for pregnancy.

Key result

Dose descriptor:

dose level: Highest dose for the preliminary study.

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

not examined

Changes in sex ratio:

not examined

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

not examined

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined

Key result

Remarks on result:

not measured/tested

Key result

Abnormalities:

not examined

Key result

Developmental effects observed:

not specified

Staircase phase: Group 1: non pregnant females.

- General condition: The general condition of the females was unaffected by treatment and no deaths occurred. Salivation was recorded on number of occasions for a short period immediately after dosing. The frequency of recorded salivation was increased at dosages of 1000 and 2000 mg/kg bw/day.

- Bodyweights:

Bodyweight and bodyweight gain in the non-pregnant female was essentially unaffected by treatment with LAE at dosages of up to 2000 mg/kg bw/day. At the highest dose (2000 mg//kg/day) three of the four animals showed minor losses in bodyweight after the first dose but weight gain was recovered the following day.

- Necropsy findings: No adverse findings were recorded at necropsy of the non-pregnant females after a total of 8 doses of LAE which escalated from 250 to 2000 mg/kg bw/day.

Constant dosage phase: Group 2: pregnant females.

- General condition: The general condition of the females was unaffected by treatment and no deaths occurred. Salivation was recorded occasionally for all animals for a short period immediately after dosing toward the end of the treatment period.

- Bodyweights: Bodyweight and cumulative bodyweight gain in the pregnant female appeared to be unaffected by treatment with LAE at a dosage of 2000 mg/kg bw/day.

- Necropsy findings: All females were pregnant at termination and no adverse findings were recorded at necropsy of the pregnant females after a total of 7 doses of LAE at 2000 mg/kg bw/day.

Conclusions:

The objective of this tolerance study was to assess the effects of repeated oral administration of Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE, also known as Lauramide of ethyl arginate) to non-pregnant and pregnant rats in order to establish the maximum dosage for use in a preliminary study of embryo-fetal development. After results' evaluation, the highest dosage for use in a preliminary embryo-fetal study in the rat should be 2000 mg/kg bw/day (the highest dosage that could be achieved for this species without exceeding without exceeding guideline figures for volume dosage).

Executive summary:

The objective of this tolerance study was to assess the effects of repeated oral administration of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE,also known as Lauramide of ethyl arginate) to non-pregnant and pregnant rats in order to establish the maximum dosage for use in a preliminary study of embryo-fetal development.

LAE was administered by gavage to four non-pregnant rats (Charles River Crl:CD® BR rats), commencing at an initial dosage of 250 mg/kg bw/day for two days. In the absence of any adverse response, the dosage was doubled every two days until reaching a dosage of 2000 mg/kg bw/day, which was the maximum practical dosage based on the concentration of the formulation suspension and a volume of 10 ml/kg. Four females were then mated and received LAE at a dosage of 2000 mg/kg bw/day for seven consecutive days from Day 6 of gestation.

Following completion of treatment, all animals were killed and examined for evidence of reaction to treatment.

 

1.- Staircase phase (Group 1 : escalating dosages from 250 to 2000 mg/kg bw/day)

The general condition, clinical signs and bodyweights of non-pregnant females were not significantly affected by treatment at dosages of up to 2000 mg/kg/day and there were no adverse findings at necropsy which were considered to be treatment related.

 

2. - Constant dosage phase (Group 2 :2000 mg/kg bw/day)

The general condition, clinical signs and bodyweights of pregnant females were unaffected by treatment at 2000 mg/kg/day and there were no adverse findings at necropsy which were considered to be related to treatment. Embryo survived to Day 13 of gestation was not compromised.

Oral administration of LAE to non-pregnant rats at dosages between 250 and 2000 mg/kg bw/day produced no significant adverse findings. The occurrence of salivation immediately after oral administration of test materials is not unusual and is not considered torepresent a significant reaction to treatment.

Subsequent treatment of pregnant rats at 2000 mg/kg bw/day for 7 consecutive days did not produce any significant disturbance of maternal bodyweight or embryo survival.

It was concluded from this investigation that the highest dosage for use in a preliminary embryo-fetal study in the rat should be 2000 mg/kg bw/day (the highest dosage that could be achieved for this species without exceeding guideline figures for volume dosage).

Reference 5

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 23, 1998 to August 6, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: Tolerance study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: approximately 10 to 11 weeks of age
- Weight at study initiation: 196 to 269 g
- Housing: Rats were housed in TR18 cages from Arrowmight Biosciences, Hereford, England and RB3 modified cages from North Kent Plastic Cages Limited, Erith, Kent, England. The cages consisted of stainless steel (TR18) or high density polypropylene (RB3) bodies with lids and floors of stainless steel grid and were suspended in batteries over trays covered with absorbent paper which was replaced twice weekly or daily during pairing.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum of 10 days acclimatisation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-25°C
- Humidity (%): 40-70%
- Air changes (per hr): 15 room air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light: 12-hour dark cycle

IN-LIFE DATES: From: February 18, 1998 To: March 26, 1998

Route of administration:

oral: gavage

Vehicle:

other: Methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): freshly each day

VEHICLE
- Justification for use and choice of vehicle (if other than water): Methylcellulose
- Concentration in vehicle: suspension in 1% Methylcellulose

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Information on the homogeneity of mixing, stability and concentration of the test material in the vehicle was determined. Specimen formulations of 600 ml were prepared at each of the highest and lowest concentrations. These were then separated into 3 x 200ml aliquots for each concentration. Aliquot 1 was stored at 21°C and analysed at one, two, four and 48 hours; aliquots 2 and 3 were stored at 4°C and analysed at 48 hours and 8 days respectively.

Details on mating procedure:

Females were paired on a one-to-one basis with stock males of the same strain. Each morning following pairing, the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each female and examined for the presence of spermatozoa.
The day on which a sperm positive vaginal smear or at least three copulation plugs were found was designated Day 0 of gestation.

Duration of treatment / exposure:

From Day 6 to 19 of gestation.

Frequency of treatment:

Daily to each animal based upon the animal's bodyweight on that day

Duration of test:

From April 17, 1998 to May 3, 1998

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

200 mg/kg bw/day

Dose / conc.:

600 mg/kg bw/day

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

6 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages of 200, 600 and 2000 mg/kg bw/day were based on results from a tolerance study performed at these laboratories. In that study 2000 mg/kg bw/day was considered to the highest dosage that could be administered to the rat on a daily basis without exceeding guideline figures for volume dosage.
- Rationale for animal assignment (if not random): Rat was chosen because it satisfies the requirement for a rodent species by regulatory agencies. The Charles River Crl: CD® BR rat strain was used because of the historical control data available in this laboratory.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice daily throughout the study and any visible signs of reaction to treatment were recorded, with details of type, severity, time of onset and duration.

BODY WEIGHT: Yes
- Time schedule for examinations: Females were weighed on Days 0, 3 and 6 to 20 inclusive after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was recorded for the periods Days 0-2, 3-5, 6-8, 9-11, 12-15, 16-17 and 18-19 after mating.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. For each animal, mean daily intakes were calculated for periods of Days 0-2, 3-5, 6-8, 9-11, 12-15, 16¬17 and 18-19 of gestation. Group mean daily intakes and SD were calculated for these periods.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Each animal was first examined macroscopically for evidence of disease or adverse reaction to treatment and specimens of abnormal tissues were retained.

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

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: No data
- Head examinations: No data

Statistics:

The small sample size in this study precluded meaningful statistical evaluation. Inter-group differences were assessed by reference to control data previously recorded in these laboratories.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The general condition of the animals was satisfactory and 21 of the 23 females surviving to the end of the study were pregnant. For simplicity, one female in the Control group has been termed “not pregnant” and excluded from group mean values although staining the uterus revealed a single implantation site. One female (F7) receiving 200 mg/kg/day was killed in extremis on Day 19 of gestation after showing reduced food intake on Days 18-19 of gestation, (only 2g/day) and bodyweight loss from Day 18 to 19 o f 40g. This female had signs of pallor, piloerection, brown staining around left orbital, red urine and a perigenital discharge at despatch. Necropsy revealed a large amount of dark red fluid within the vagina and both uterine horns. The uterus contained 15 late resorptions. Salivation after dosing was occasionally seen in 3 of the 6 animals receiving 600 mg/kg/day and on about 50% of occasions in all animals receiving 2000 mg/kg/day. One female in each of the treated groups had periods when respiration sounded noisy. There were no other significant clinical signs recorded in either the control group or any of the treatment groups,

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One femalle (F7) was killed in extremis.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no intergroup differences in bodyweight or bodyweight gain which were considered to be treatment-related. Occasional animals in all groups showed slight bodyweight loss during the first two days of treatment but this was considered to be related to animals adapting to the dosing process rather than to the test material itself.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was similar for all groups of animals throughout the treatment period, apart from the one female (F7) at 200 mg/kg/day which showed slightly reduced food intake during days 16-17 and then virtually stopped eating.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no necropsy findings which were considered to be related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

not examined

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

One control female (F1) had only one single implantation site, revealed by staining the uterus, and has been excluded from group mean values. Rats with very low implantation rates frequently show spontaneous resorption at an early stage of pregnancy. Another two animals, one in each of the groups receiving 200 mg/kg/day and 2000 mg/kg/day showed high pre-implantation losses, but because these losses almost certainly occurred before the start of treatment these individual incidences arc considered not to be treatment related. The group mean value for post-implantation loss was slightly higher in animals receiving 600 mg/kg/day than other groups, but in the absence of similar effect in the highest dosage group this was considered to be unrelated to treatment.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

not examined

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

not specified

Other effects:

no effects observed

Key result

Dose descriptor:

dose level: Highest dosage for the teratology study.

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no obvious treatment related effects upon fetal development as assessed by fetal weight and macroscopic examination at necropsy.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified

Reduction in number of live offspring:

not specified

Changes in sex ratio:

not specified

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Description (incidence and severity):

There were no obvious treatment related effects upon fetal development as assessed by fetal weight and macroscopic examination at necropsy.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Key result

Dose descriptor:

dose level: Highest dosage for the teratology study.

Effect level:

2 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Fetal weight and macroscopic examination at necropsy.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

The results of the analyses of the concentration of test material in formulations confirm acceptable homogeneity and stability and that concentrations of analysed preparations from the first and last weeks of treatment were satisfactory.

Conclusions:

Assessment of influence on progress and outcome of pregnancy in CD rats in order to establish suitable dosages for a main embryo-fetal toxicity study of Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) by oral gavage administration. 2000 mg/kg bw/day of LAE would be suitable as the highest dose level for a main embryo-fetal study in the rat.

Executive summary:

In this preliminary study, the influence of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) study,was assessed in sexually mature rats of the CD strain, to establish suitable dosages for a main embryo-fetal toxicity study.

 

The oral route was selected to simulate the conditions of possible human exposure. Dosages of 200, 600 and 2000 mg/kg bw/day were based on results from a tolerance study. In that study 2000 mg/kg bw/day was considered to the highest dosage that could be administered to the rat on a daily basis without exceeding guideline figures for volume dosage.

 

For this purpose, LAE was administered by gavage at dosages of 200, 600 or 2000 mg/kg bw/day to groups of 6 presumed pregnant rats from Day 6 to 19 after mating, inclusive. Control animals received the vehicle, 1% w/v Methylcellulose, throughout the same period.

 

Surviving females were killed on Day 20 after mating for examination of their uterine contents. Results

 

There were no significant treatment related effects on females receiving LAE at 200, 600 or 2000 mg/kg bw/day from Day 6 of gestation until just before the expected time of parturition, or upon the survival or development of their fetuses. Salivation, seen after dosing particularly in the highest dosage group, is a common non-specific response to gavage treatment which may relate to the taste or pH of the test material. It usually has no toxicological significance.

There were no obvious treatment related effects upon fetal survival or fetal development as assessed numbers of live fetuses, fetal weight and macroscopic examination of the fetuses at necropsy.

 

It was, therefore, concluded that 2000 mg/kg bw/day of LAE would be suitable as the highest dose level for a main embryo-fetal study in the rat. This dosage is considered to be the maximum achievable dosage in relation to formulation and volume dosage under the condition of use.

 

Reference 6

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 6, 1998 to August 6, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: 18-26 weeks old on arrival
- Weight at study initiation: 3.46 to 4.08 Kg
- Housing: Singly in suspended plastic cages (type LCP 3/4) mounted in batteries (Lab Care Precision Limited, England). All animals were kept on the same battery to minimise, as far as possible, environmental influences. The cages had a floor area of 5400 sqcm and were 45 cm high and were fitted with perforated counter-sunk floor panels. An undertray beneath the floor was lined with absorbent crepe-paper which was changed at least three times per week.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum to three week's acclimatisation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-23°C
- Humidity (%): 40-70%
- Air changes (per hr): approximately 17 to 20 room air changes per hour.
- Photoperiod (hrs dark / hrs light): 14-hour light: 10-hour dark cycle operated throughout

IN-LIFE DATES: From: March 11, 1998 To: April 6, 1998

Route of administration:

oral: gavage

Vehicle:

other: Methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): freshly each day

VEHICLE
- Justification for use and choice of vehicle (if other than water): Methylcellulose
- Concentration in vehicle: 1% w/v Methylcellulose

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Females were naturally mated with New Zealand White males of established fertility. Following mating, each female was injected intravenously with 25 i.u. of luteinising hormone (Profasi, Serono), to ensure successful ovulation. The day of mating was designated Day 0 of gestation

Duration of treatment / exposure:

Group 1:
Day 1-2: 60 mg/kg/day
Day 3-4: 120 mg/kg/day
Day 5-6: 250 mg/kg/day
Day 7-8: 500 mg/kg/day
Day 9-10: 1000 mg/kg/day

Group 2:
From Day 6 to 12 of gestation: 1000 mg/kg/day

Frequency of treatment:

Daily to each animal based upon the animal's bodyweight on that day

Duration of test:

From March 11, 1998 to April 6, 1998

Dose / conc.:

60 mg/kg bw/day

Remarks:

Staircase phase (group 1)

Dose / conc.:

120 mg/kg bw/day

Remarks:

Staircase phase (group 1)

Dose / conc.:

250 mg/kg bw/day

Remarks:

Staircase phase (group 1)

Dose / conc.:

500 mg/kg bw/day

Remarks:

Staircase phase (group 1)

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Staircase phase (group 1)

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Constant dosage phase (group 2)

No. of animals per sex per dose:

Staircase phase: 2 females.
Constant dosage phase: 2 females.

Control animals:

no

Details on study design:

STUDY SCHEDULE
1. Staircase phase in non-pregnant females:
Two non-pregnant females were allocated to this phase of the investigation. Treatment commenced at an initial dosage of 60 mg/kg bw/day for two days. For subsequent two-day periods the dosage was doubled (approximately) until reaching the maximum practical dosage of 1000 mg/kg bw/day (based on the physical properties of the formulated suspension and the volume dosage).

Dosages received on the various days of the study were as follows:

Day of treatment 1-2 3-4 5-6 7-8 9-10
Dosage (mg/kg/day) 60 120 250 500 1000

2. Constant dosage phase in pregnant females
Following consultation with the Sponsor, two females were mated with proven males and dosed on Days 6 to 12 of gestation inclusive at 1000 mg/kg bw/day. Animals were terminated on Day 13 of gestation.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes.
All animals were weighed and examined daily throughout the study and any visible signs of reaction to treatment were recorded with details of type, severity, time of onset and duration.

BODY WEIGHT: Yes
Time schedule for examinations: All animals were weighed and examined daily throughout the study and any visible signs of reaction to treatment were recorded with details of type, severity, time of onset and duration.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data

TERMINAL STUDIES:Yes
Each animal was examined macroscopically for evidence of disease or adverse reaction to treatment and specimens of tissues considered abnormal were retained in an appropriate fixative. Inseminated animals were checked for pregnancy.

Clinical signs:

no effects observed

Description (incidence and severity):

Staircase phase: Group 1 : non-pregnant females
The general conditon of the animals was unnafected by treatment.

Constant dosage phase: Group 2: pregnant females
All animals survived 7 days of treatment and were killed on Day 13 of pregnancy. Both females showed reduced food and water intake early in the treatment period (Day 8-9 of gestation). The effect on water intake was transient but reduced food consumption and reduced faecal output were observed daily until termination. On Day 7 of gestation one animal (F3) became stressed during dosing on the second day of treatment and dosing had to be delayed by 30 minutes and performed later. On Day 8 of gestation, the second animal (F4) showed marked respiratory noises leading to irregular respiration, blue extremities, innactivity and hunched posture within an hour after receiving its third dose. The more severe signs lasted until the end of the day and noisy respiration was recorded daily until termination.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

Staircase phase: Group 1 : non-pregnant females
No deaths occured.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Staircase phase: Group 1 : non-pregnant females
Bodyweight and bodyweight gain in the non-pregnant female was essentially unaffected by treatment with LAE at dosages of up to 1000 mg/kg/day. Marginal losses in bodyweight were recorded for one of the females on one day at 500 mg/kg/day and for both females at 1000 mg/kg/day on one day only.

Constant dosage phase: Group 2: pregnant females
Weight loss, in excess of 400 g per animal, was recorded for both animals by Day 10 of gestation . One animal (F3) continued to lose weight to termination on Day 13 of gestation, but the other female (F4), which had shown the respiratory problems, actually gained small amounts of bodyweight between Day 10 and Day 13 of gestation.

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

Constant dosage phase: Group 2: pregnant females
All animals survived 7 days of treatment and were killed on Day 13 of pregnancy. Both females showed reduced food and water intake early in the treatment period (Day 8-9 of gestation). The effect on water intake was transient but reduced food consumption and reduced faecal output were observed daily until termination. On Day 7 of gestation one animal (F3) became stressed during dosing on the second day of treatment and dosing had to be delayed by 30 minutes and performed later. On Day 8 of gestation, the second animal (F4) showed marked respiratory noises leading to irregular respiration, blue extremities, innactivity and hunched posture within an hour after receiving its third dose. The more severe signs lasted until the end of the day and noisy respiration was recorded daily until termination.

Food efficiency:

not examined

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

not specified

Description (incidence and severity):

Constant dosage phase: Group 2: pregnant females
All animals survived 7 days of treatment and were killed on Day 13 of pregnancy. Both females showed reduced food and water intake early in the treatment period (Day 8-9 of gestation). The effect on water intake was transient but reduced food consumption and reduced faecal output were observed daily until termination. On Day 7 of gestation one animal (F3) became stressed during dosing on the second day of treatment and dosing had to be delayed by 30 minutes and performed later. On Day 8 of gestation, the second animal (F4) showed marked respiratory noises leading to irregular respiration, blue extremities, innactivity and hunched posture within an hour after receiving its third dose. The more severe signs lasted until the end of the day and noisy respiration was recorded daily until termination

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Description (incidence and severity):

Staircase phase: Group 1 : non-pregnant females
No adverse findings were recorded at necropsy of the non-pregnant females after a total of 10 doses of LAE which were progressively increased from 60 to 1000 mg/kg/day.

Constant dosage phase: Group 2: pregnant females
Both females were pregnant at termination and there were no apparent adverse findings on the conceptus. Both animals showed some evidence of collapse of the areas of the lung and these findings were more extensive and accompanied by suggestions of infection in the lungs of the animal which had shown signs of respiratory distress during the course of treatment. Both animals showed prominent dark vessels on the surface of the kidneys, but the significance of this observation was uncertain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

not examined

Pre- and post-implantation loss:

not examined

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

not examined

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

not examined

Other effects:

no effects observed

Key result

Dose descriptor:

dose level: Highest dosafe for the preliminary teratology study.

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

not specified

Description (incidence and severity):

It is recommended that the highest dosage for the preliminary teratology study should be 1000 mg/kg/day. This will permit investigation of the effects of LAE on the pregnant rabbit in the preliminary embryo-fetal study at a dosage which is commonly accepted as a maximum limit dosage in studies of this type.

Fetal body weight changes:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

not examined

Changes in sex ratio:

not examined

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

not examined

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined

Key result

Remarks on result:

not measured/tested

Key result

Abnormalities:

not examined

Key result

Developmental effects observed:

not specified

Staircase phase: Group 1: non-pregnant females:

- General condition: The general condition of the females was unaffected by treatment and no deaths occurred.

No overt dosage related signs were recorded.

- Bodyweights: Bodyweight and bodyweight gain in the non-pregnant female was essentially unaffected by treatment with LAE at dosages of up to 1000 mg/kg/day. Marginal losses in bodyweight were recorded for one of the females on one day at 500 mg/kg bw/day and forboth females at 1000 mg/kg/day on one day only.

- Necropsy findings: No adverse findings were recorded at necropsy of the non-pregnant females after a total of 10 doses of LAE which were progressively increased from 60 to 1000 mg/kg bw/day.

 

Constant dosage phase: Group 2: pregnant females:

- General condition: All animals survived 7 days of treatment and were killed on Day 13 of pregnancy.

Both females showed reduced food and water intake early in the treatment period (Day 8-9 of gestation).

The effect on water intake was transient but reduced food consumption and reduced faecal output were observed daily until termination.

 

- Bodyweights: Weight loss, in excess of 400 g per animal, was recorded for both animals by Day 10 of gestation. One animal (F3) continued to lose weight to termination on Day 13 of gestation, but the other female (F4), which had shown the respiratory problems, actually gained small amounts of bodyweight between Day 10 and Day 13 of gestation.

 

- Necropsy findings: Both females were pregnant at termination and there were no apparent adverse findings on the conccptus. Both animals showed some evidence of collapse of the areas of the lung and these findings were more extensive and accompanied by suggestions of infection in the lungs of the animal which had shown signs of respiratory distress during the course of treatment. Both animals showed prominent dark vessels on the surface of the kidneys, but the significance of this observation was uncertain.

Conclusions:

Oral administration of Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) to non-pregnant rabbits at dosages between 60 and 1000 mg/kg bw/day as a series of escalating doses for two days at each dose level had no apparent adverse effects upon the animals. Treatment at 1000 mg/kg bw/day for 7 consecutive days did not result in any significant effect on embryo survival.

Executive summary:

The objective of this tolerance study was to assess the effects of repeated oral administration of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE,also known as Lauramide of ethyl arginate) to non-pregnant and pregnant rabbits in order to establish the maximum suitable dosage for use in a preliminary study of embryo-fetal development. The oral route was selected to accord with the possible route of human exposure; administration by gavage is conventionally used in embryo-fetal studies where precise control of dosages is desirable during the organogenesis phase of pregnancy.

Oral administration of LAE to non-pregnant rabbits at dosages between 60 and 1000 mg/kg bw/day as a series of escalating doses for two days at each dose level had no apparent adverse effects upon the animals.

A dose of 1000 mg/kg/day was administered to pregnant rabbits from Day 6 of gestation. There were apparent effects upon food and water intake from the second or third dose resulting in reduced faecal pellet production and loss in bodyweight. These signs are common indicators of disturbance to the equilibrium of the rabbit and may indicate a specific response to treatment. However, interpretation is complicated in this study by the occurrence of respiratory distress in one animal and necropsy evidence of lung damage in both animals. The lung findings wereconsidered unlikely to be related to toxicity of the test material but may relate to stress or to aspiration of small amounts of the dosing suspension, although there was no overt evidence of formulated material within the lungs at necropsy.

Despite maternal findings, treatment at 1000 mg/kg bw/day for 7 consecutive days did not result in any significant effect on embryo survival.

In view of the complication with possible non-treatment related changes in the lungs it is recommended that the highest dosage for the preliminary teratology study should be 1000 mg/kg bw/day. This would ensure that the effects of LAE on the pregnant rabbit were investigated at a dosage which is commonly accepted as a maximum limit dosage in studies of this type. Moreover, there was no evidence of the expected loose faeces which are often a typical reaction by rabbits to antibiotics which kill the gut flora and disturb the normal nutritional pattern of the rabbit and which might preclude further investigations at such a high dosage.

Reference 7

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 23, 1998 to August 6, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

other: Tolerance study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: approximately 18 to 26 weeks old
- Weight at study initiation: 2.93 to 4.07 kg at arrival; 3.11 kg to 4.26 kg on day 6 after mating
- Housing: Singly in suspended anodiscd aluminium cages (Type TR6) mounted in batteries (NKP Cages LTD, Erith, Kent, England) and were randomly distributed on the battery in order to equalise, as far as possible, environmental influences among the groups. The cages were fitted with perforated counter sunk floor panels, and an undertray beneath the floor was lined with absorbent paper, which was changed at least three times per week.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum of 7 days acclimatisation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-23°C
- Humidity (%): 40-70%
- Air changes (per hr): 12 room air changes per hour
- Photoperiod (hrs dark / hrs light): a 14-hour light: 10-hour dark cycle throughout the study

IN-LIFE DATES: From: May 18, 1998 To: June 17, 1998

Route of administration:

oral: gavage

Vehicle:

other: Methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The animals were dosed daily from Day 6 to 19 of gestation inclusive at a volume-dosage of 5 ml/kg bw/day. Control animals received the vehicle at the same volume-dosage during the treatment period. The volume administered daily to each animal was based on the most recently recorded bodyweight.
DIET PREPARATION
- Rate of preparation of diet (frequency): freshly each day
VEHICLE
- Justification for use and choice of vehicle (if other than water): Methylcellulose
- Concentration in vehicle: suspension in 1% Methylcellulose

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of test material in formulations was used in this study was analysed on one day for each of the first and last weeks of dosing. Four 2 ml samples were taken from each group and two samples for each group were analysed for test material content. Remaining samples were stored at approximately -20°C pending possible requirement for re-analysis before being discarded following satisfactory completion of the study.

Details on mating procedure:

Females were time-mated with New Zealand White males of established fertility at Charles River U.K. Ltd. The day of mating was designated Day 0 of gestation. Each female was identified by a unique animal number in the ear and records of parentage and the identity of the male partner were supplied.

Duration of treatment / exposure:

From Day 6 to 19 of gestation.

Frequency of treatment:

The animals were dosed daily by the oral route (gavage) from Day 6 to 19 of gestation inclusive at a volume-dosage of 5 ml/kg bw/day. Control animals received the vehicle at the same volume-dosage during the treatment period. The volume administered daily to each animal was based on the most recently recorded bodyweight.

Duration of test:

From May 18, 1998 to June 17, 1998

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

Group 1 (Control, 0 mg/kg bw/day): 6 females
Group 2 (250 mg/kg bw /day): 4 females
Group 3 (500 mg/kg bw/day): 4 females
Group 4 (1000 mg/kg bw/day): 4 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages of 250, 500 and 1000 mg/kg bw/day were based on results from a tolerance study, where administration of 1000 mg/kg bw/day for up to seven consecutive days was associated with slight disturbance of food intake and bodyweight gain. The 14 day dosing period was chosen to encompass the period of major organogenesis.
- Rationale for animal assignment (if not random): The rabbit was chosen because it satisfies the requirement for a non rodent species by regulatory agencies. The New Zealand White strain was used because of the historical control data available.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice daily throughout the study and any visible signs of reaction to treatment were recorded, with details of type, severity, time of onset and duration.

BODY WEIGHT: Yes
- Time schedule for examinations: Females were weighed daily throughout the study and bodyweights are reported for Days 1, 3, 6, 8, 10,12,14,16,18, 20, 24 and 28 after mating. Bodyweights recorded by the supplier after mating (Day 0) and weights on intermediate days arc retained with the raw data.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was recorded for the periods 1-5, 6-12, 13-19, 20-23, and 24-28 inclusive after mating.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. For each animal, mean daily intakes were calculated for periods of Days 1-5, 6-12, 13-19, 20-23 and 24-28 of gestation. Group mean daily intakes and SD were calculated for these periods.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: Each animal was first examined macroscopically for evidence of disease or adverse reaction to treatment and specimens of abnormal tissues were retained.

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

Fetal examinations:

- External examinations: Yes. Individual placental weights and placental abnormalities were recorded.
- Soft tissue examinations: Yes. The neck, thoracic and abdominal cavities of all fetuses from each litter were dissected and examined, and the sex of each fetus recorded and position within the litter recorded. Following examination the fetuses were eviscerated and one third of the fetuses in each litter were decapitated and the heads fixed in Bouins fluid. Torsos and the remaining intact fetuses were fixed in industrial methylated spirits.
- Skeletal examinations: No data
- Head examinations: Yes

Statistics:

The small sample size in this study precluded meaningful statistical evaluation. Inter-group differences were assessed by reference to control data previously recorded in these laboratories. For presentation purposes the values shown in appendices may be rounded. For the calculation of group mean values, unrounded values may have been used and therefore it may not always be possible to calculate these exactly by using the values presented in the appendices.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All animals allocated to the study were pregnant and had live fetuses at necropsy on Day 29 of gestation. One animal at 250 mg/kg/day and one at 1000 mg/kg/day had periods when the respiration was noisy and/or slow but this did not appear to be dosage related and there were no other remarkable clinical signs recorded in either the Control group or any of the treatment groups

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

not specified

Description (incidence and severity):

The majority of females showed good bodyweight gain between Day 1 and Day 6 of pregnancy while acclimatising to laboratory conditions before treatment started. Small losses in bodyweight were recorded between Day 6 and Day 12 of gestation (the first week of treatment) for two of six control animals, one of four animals at 250 , two of four animals at 500 mg/kg/day and three of four animals 1000 mg/kg/day and other animals showed brief periods of weight loss. Thereafter most animals gained bodyweight, and by Day 28 of pregnancy group mean bodyweight gains were similar in the control and low dose group and only marginally low in the intermediate and high dose groups.
Adjusted bodyweight gains (calculated by subtracting the weight of the gravid uterus from the weight gain between Day 6 and Day 29) showed that there were no intergroup differences and that all females had lost weight to the developing conceptus during pregnancy, a findings which is normal for this strain of rabbit.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption by animals receiving 1000 mg/kg/day was lower than that of the Control group during the period Days 6-12 of gestation and was slightly low for the rest of the treatment period; intake was slightly depressed for females receiving 500 mg/kg/day throughout treatment. In the first four days after treatment stopped, animals that had received 500 or 1000 mg/kg/day showed increased food consumption relative to consumption during the treatment period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no necropsy findings which were considered to be related to treatment for the treated females at the end of pregnancy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

There were no apparent treatment related effects on embryo-fetal survival

Pre- and post-implantation loss:

not specified

Description (incidence and severity):

The numbers of corpora lutea were essentially similar in all groups but intergroup variation in pre-implantation loss (occurring before treatment started) and slightly high levels of post-implantation loss at 500 mg/kg/day resulted in considerable differences in mean live litter size.

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Overall fetal weight was lowest at 1000 mg/kg/day but this was largely attributable to the effect of larger litter size in this group and there was no indication that the ability of the dam to support a litter was impaired by treatment. There was a low incidence of fetal anomalies seen at necropsy but no indication of any adverse effect of treatment.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Two animals, one in each of the groups receiving 250 and 1000 mg/kg bw/day showed periods of respiratory distress during the treatment phase of the study. Similar observations have been made in other studies performed with this test material in rabbits and in rats but the nature of the reaction has not been defined. Reduced food consumption during treatment at 500 and at 1000 mg/kg bw/day, leading to slight losses in bodyweight were the only effects considered to be clearly related to treatment.

Key result

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

not specified

Description (incidence and severity):

Reduced food consumption leading to slight losses in bodyweight gain.

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Overall fetal weight was lowest at 1000 mg/kg/day but this was largely attributable to the effect of larger litter size in this group and there was no indication that the ability of the dam to support a litter was impaired by treatment.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no apparent treatment related effects on embryo-fetal survival.

Changes in sex ratio:

not specified

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

The numbers of corpora lutea were essentially similar in all groups but intergroup variation in pre-implantation loss (occurring before treatment started) and slightly high levels of post-implantation loss at 500 mg/kg/day resulted in considerable differences in mean live litter size.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

There were no apparent treatment related effects on embryo-fetal survival.

External malformations:

no effects observed

Description (incidence and severity):

There was a low incidence of fetal anomalies seen at necropsy but no indication of any adverse effect of treatment.

Skeletal malformations:

not specified

Visceral malformations:

not specified

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no obvious treatment related effects upon fetal development as assessed by fetal weight and macroscopic examination at necropsy. There were no apparent treatment related effects on embryo-fetal survival. The numbers of corpora lutea were essentially similar in all groups but intergroup variation in pre-implantation loss (occurring before treatment started) and slightly high levels of post-implantation loss at 500 mg/kg bw/day resulted in considerable differences in mean live litter size.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: fetotoxicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

The influence of Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) upon the progress and outcome of pregnancy was assessed in sexually mature rabbits of the New Zealand White strain, to establish suitable dosages for a main embryo-fetal toxicity study. On the basis of this preliminary study 250 mg/kg bw/day was considered to be the no-effect-level (NOEL) for the mother, but both 500 and 1000 mg/kg bw/day were associated with reduced food consumption and reduced bodyweight gain. 1000 mg/kg bw/day wasconsidered to be the NOEL for the fetus. A dosage of up to 1000 mg/kg bw/day would be suitable as the highest dosage level for a main embryo-fetal study in the rabbit.

Executive summary:

The objective of this preliminary study was to assess the effects of repeated oral administration of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE), upon the progress and outcome of pregnancy in the rabbit in order to establish suitable dosages for a main embryo-fetal toxicity study in the rabbit. The influence of LAE upon the progress and outcome of pregnancy was assessed in sexually mature rabbits of the New Zealand White strain, to establish suitable dosages for a main embryo-fetal toxicity study.

For this purpose, LAE was administered by gavage at dosages of 250, 500 or 1000 mg/kg/day to groups of 4 presumed pregnant rabbits from Day 6 to 19 after mating, inclusive. Six females designated as Control animals received the vehicle, 1% w/v Methylcellulose, throughout the same period. All females were killed on Day 29 after mating for examination of their uterine contents.

All animals allocated to the study were pregnant and had live fetuses at necropsy on Day 29 of gestation. One animal at 250 mg/kg bw/day and one at 1000 mg/kg bw/day had periods when the respiration was noisy and/or slow but there were no other clinical signs of importance.

 

Small losses in bodyweight were recorded during the first week of treatment (Day 6 to 12 of gestation) for three of four animals at 1000 mg/kg bw/day and for a lower proportion of the Control, low and intermediate dosage groups. Weight gain by the completion of treatment was still low at 500 and 1000 mg/kg/day compared to Controlbut there were no meaningful intergroup differences in maternal weight gain by the end of pregnancy.

 

Food consumption by animals receiving 1000 mg/kg bw/day was lower than that of the Control group during the period Days 6-12 of gestation and remained slightly low to the end of the treatment period, but was then higher than Control in the immediate post-treatment phase. Animals receiving 500 mg/kg/day showed slightly low food consumption during treatment with an increase after completion of dosing. No effects were seen at 250 mg/kg bw/day.

 

There were no necropsy findings among the treated females and no effects on fetal survival. Overall fetal weight was lowest at 1000 mg/kg bw/day but this was largely attributable to the effect of larger litter size in this group. There was a low incidence of fetal anomalies seen at necropsy but no indication of any adverse effect of treatment.

Two animals, one in each of the groups receiving 250 and 1000 mg/kg bw/day showed periods of respiratory distress during the treatment phase of the study. Similar observations have been made in other studies performed with this test material in rabbits and in rats but the nature of the reaction has not been defined. Reduced food consumption during treatment at 500 and at 1000 mg/kg bw/day, leading to slight losses in bodyweight were the only effects considered to be clearly related to treatment.

 

On the basis of this preliminary study 250 mg/kg/day was considered to be the no-effect-level (NOEL) for the mother, but both 500 and 1000 mg/kg/day were associated with reduced food consumption and reduced bodyweight gain. 1000 mg/kg bw/day was considered to be the NOEL for the fetus. A dosage of up to 1000 mg/kg bw/day would be suitable as the highest dosage level for a main embryo-fetal study in the rabbit.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Study duration:

subacute

Species:

other: rats and rabbits

Quality of whole database:

Klimisch 1. The studies were carried out in accordance with internationally valid GLP principles.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Observations and results obtained under the Guidelines mentioned show that dehydrated Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) revealed that the No Adverse Effect Level (NOAEL) for Developmental toxicity performance in rats and rabbits for the dam was 200 but 2000 mg/kg bw/day was the NOAEL for the fetuses of dams that survived to the end of pregnancy under the experimental conditions described.

Justification for classification or non-classification

Results from reports assessed, revealed that No Adverse Effect Level (NOAEL) for reproductive performance in the CD rat is 15000 ppm LAE equivalent to at least 1073 mg/kg bw/day.

Results from the developmental toxicity studies showed that the NOAEL for the fetus was the highest tested dose for both rat (2000 mg/kg bw/day) and rabbit (1000 mg/kg bw/day).

According to CLP Regulation and based on the available data, the substance is not classified for toxicity to reproduction.