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

Toxicity to reproduction

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

434-630-6

EC Name:

-

Cas Number:

60372-77-2

Molecular formula:

Hill formula: C20H41N4O3Cl

IUPAC Name:

ethyl N2-dodecanoyl-l-argininate hydrochloride

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: Nα-Lauroyl-L-arginine ethyl ester monohydrochloride
- Physical state: White powder
- Storage condition of test material: In a refrigerator at approximately 4 ° C
- Lot/batch No.: 10234
- Expiration date of the lot/batch: March 19, 2004
- Purity: 88.2%

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

2 500 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

6 000 ppm (nominal)

Doses / concentrations 4

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.

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

1

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)

2

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)

3

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)

4

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)

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Results: F1 generation

General toxicity (F1)

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

Effect levels (F1)

1

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)

2

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)

3

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)

4

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)

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

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.

Developmental neurotoxicity (F2)

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 (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

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.

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

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.

 

Applicant's summary and conclusion

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.