Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
yes
Remarks:
Small deviations that were considered to not have affected the validity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
L-isoleucine
EC Number:
200-798-2
EC Name:
L-isoleucine
Cas Number:
73-32-5
Molecular formula:
C6H13NO2
IUPAC Name:
L-isoleucine
Test material form:
solid: crystalline
Specific details on test material used for the study:
- Name of test material: L-isoleucine -- feed grade
- Physical state: white, crystalline powder
- Analytical purity: 97.2%
- Lot/batch No.: Batch production june 2006
- Expiration date of the lot/batch: 1 July 2008
- Storage condition of test material: ambient temperature, in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Species: Wistar outbred Crl:WI(WU)
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 4 weeks
- Weight at study initiation: body weight variation did not exceed 20%
- Fasting period before study: not specified
- Housing: macrolon cages with wood shavings (Lignocel, Type 3/4) as bedding material and strips of paper (Enviro-dri) as environmental enrichment
- Diet (e.g. ad libitum): cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham, England); ad libitum
- Water (e.g. ad libitum): tap water suitable for human consumption; ad libitum
- Acclimation period: 12 days (including 2 days of quarantine)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12/12

The temperature and relative humidity in the animal room however, exceeded the upper values indicated in the study plan at several occasions for short periods due to meteorological circumstances and/or wet cleaning of the room. Temperature reached a maximum of 24.8 °C and relative humidity was elevated up to 99.9%

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
Experimental diets were prepared by mixing powdered Rat & Mouse No. 3 breeding diet, RM3 with the appropriate amounts of test substance. The diets were mixed in a mechanical blender (Lodige, Paderbom, Germany). Fresh batches of the experimental diets were prepared on 6 occasions during the study, i.e. once monthly. The experimental diets were stored in a freezer at < -18°C.
Details on mating procedure:
During the premating period, the animals were housed in groups of 4/sex. For mating, one male and one female were housed together. Mated females were housed individually in macrolon cages, which were placed in another cage rack. The location of the mated females in the new cage racks was determined by the date of mating (females found sperm-positive on the same date were considered a "lot") and by the animal number (within each lot the mated females were housed in the order of animal number). After delivery, the cage containing the dam with litter was transferred to another rack, the location was determined by delivery date and animal number as described for mated females. After mating, the males were housed in groups of 4/sex until autopsy.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In the 13-week toxicity study (ref 1) homogeneity, stability and content were examined at dietary levels of 0.2; 1 and 5% w/w active ingredient L-isoleucine. The test substance was homogenously distributed in the diet at all dose levels and stable in the test diets when stored at room temperature for 7 days or when stored at <-18°C for 5 weeks. Since the diets prepared for this study were stored under the same conditions as in the 13-week toxicity study and dose levels were in the same range, stability and homogeneity were not determined. The content however, was analysed in diets prepared on 27 September 2006, 23 November 2006 and 12 January 2007, using the same validated analytical method as used for the supportive analyses in the 13-week toxicity study. Diet samples were extracted with an aqueous solution of trichloroacetic acid and the concentration of test substance was determined using ion chromatography after post-column derivatisation with ninhydrin reagent (amino acid analyser).
Duration of treatment / exposure:
Females were fed diets containing the test substance during a premating period of 14 weeks, during mating, gestation and lactation until sacrifice. Dams were allowed to raise one litter.

Males were fed with test diets during a premating period of 14 weeks and during mating until sacrifice.
Frequency of treatment:
Continuously, via diet.
Details on study schedule:
One generation study, no breeding was undertaken with the F1 generation.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
2%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
3%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
4%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
5%
Basis:
nominal in diet
No. of animals per sex per dose:
28 males and 28 females per dose group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The 13 week repeated dose toxicity test served as a range-finder study.
- Rationale for animal assignment (if not random): computer randomization proportionately to body weight.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

BODY WEIGHT: Yes
Body weights of male and female rats were recorded shortly before the start of the treatment at randomization and at the start of the study (premating). Males were weighed once per week during the premating and mating period until sacrifice. Females were weighed once per week during premating and mated females were weighed on days 0, 7,14 and 21 during presumed gestation and on days 1,4, 7,14 and 21 of lactation.
In addition, the animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.
Body weight changes over the different periods were calculated.

FOOD CONSUMPTION AND COMPOUND INTAKE :
Food consumption was measured per cage by weighing the feeders. Food consumption of males rats was measured weekly, except during the mating period. Food consumption of females was measured weekly during the premating period. Food consumption of mated females was recorded weekly during pregnancy and on days 1, 4, 7, 14 and 21 of lactation. The results are expressed in g per animal per day and g per kg bw per day.
Oestrous cyclicity (parental animals):
Vaginal smears were made daily in females of all groups during weeks 11,12 and 13 of the premating period to evaluate length and normality of the estrus cycle. The period of these observations was equal to that of the 13-week toxicity study. Smears were made and stained of all females, but only the smears of the control group (A) and the high-dose group (F) were evaluated.
Sperm parameters (parental animals):
Epididymal sperm motility, count and morphology
At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of 10 males of all groups. For this purpose the cauda epididymis was dissected, weighed and thereafter minced in Ml99 medium containing 0.5% bovine serum albumin. Sperm motility and, after sonification and DNA staining, the cauda epididymal sperm reserves (sperm count) were measured for all males of all groups, using the Hamilton Thome Integrated Visual Optical System (IVOS). In addition, a smear of the sperm solution was prepared and stained for all males, but only the smears of the control (group A) and the high-dose (group F) males were examined for morphology.

Testicular sperm count
At necropsy, the left testis of 19 males of all groups was placed on dry ice and subsequently stored in a freezer (< -70°C) for later determination of the number of homogenization-resistant spermatids. The testes to be analysed were thawed just before further processing. Following removal of the tunica albuginea, the testicular parenchyma were weighed, minced and homogenized in Saline Triton X-100 solution. Following DNA-staining, the homogenization-resistant sperm heads were enumerated using IVOS. The daily sperm production was calculated. The evaluation of homogenization-resistant spermatids was performed in the control group (A) and high-dose group (F).
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The total litter size and number of each sex as well as the number of stillbirths, live and dead pups and grossly malformed pups were evaluted on days 1, 4, 7, 14 and 21 of lactation. The pups were weighed individually on days 1, 4, 7, 14 and 21 of lactation. Mean pup weight was calculated per sex and both sexes combined.. The number of runts (defined as less than mean pup weight of the control group minus 2 standard deviations) was calculated.

GROSS EXAMINATION OF DEAD PUPS:
All stillborn pups and pups found dead were examined macroscopically. At weaning 1 male and 1 female pup per litter were subjected to a thorough necropsy. Pups were euthanized by exsanguination from the abdominal aorta under CO2/O2 anaesthesia and then examined grossly for pathological changes. Special attention was paid to the organs of the reproductive system.
The following organs were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde for possible future examination: brain, spleen, thymus, organs and tissues that showed macroscopic abnormalities. Brain, spleen and thymuss were weighed as soon as possible after dissection to avoid drying.

All other pups were observed for external abnormalities and sacrificed with C02/02.
Postmortem examinations (parental animals):
Ten male rats/group, also selected for sperm analysis, males that failed to sire, and all surviving female parent rats were euthanized by exsanguination from the abdominal aorta under CO2/O2 anaesthesia and then examined grossly for pathological changes. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde except for the testes which were preserved in Bouin's fixative: adrenals, brain, epididymides, kidneys, liver, ovaries*, pituitary gland*, prostate*, seminal vesicles and coagulating glands*, spleen, testes*, thyroid, uterus* (after counting of the implantation sites), vagina*, organs and tissues that showed macroscopic abnormalities.
Adrenals, brain, epididymides, kidneys, ovaries, pituitary gland, prostate, seminal vesicles and coagulating glands, spleen, testes, thyroid and uterus where weighed (paired organs together) as soon as possible after dissection to avoid drying. Tissues marked with an asterisk (*) were embedded in paraffin wax, sectioned at 5 urn, and stained with haematoxylin and eosin, except for sections of the testes, which were stained with PAS. Microscopic examination was performed on these organs of all rats of the control and high-dose groups.

All other males were examined macroscopically. In addition, reproductive organs of females that were non-mated or non-pregnant were microscopically examined.

Statistics:
The resulting data were analyzed using the methods mentioned below. Other statistical tests were performed when considered appropriate. As a level of significance was considered p<0.05.
- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test.
- Estrus cyclicity was evaluated by Fisher's exact test (number of acyclic animals and number of animals with prolonged estrus period), ANOVA followed by Dunnett's multiple comparison test (number of cycles per animal) and Kruskal-Wallis nonparametric ANOVA followed by Mann-Whitney U test (lenght of the longest cycle).
- Delivery and litter data were evaluated by Fisher's exact probability test and Kruskal-Wallis nonparametric ANOVA followed by Mann-Whitney U test
- Sperm parameters were evaluated by ANOVA followed by Dunnett multiple comparison tests (epididymal and testicular sperm count and numerical sperm motility parameters) or by Kruskal-Wallis non parametric ANOVA followed by Mann-Whitney U test (motility parameters expressed as a percentage and sperm morphology).
- Macroscopic observations and histopathological changes were evaluated by Fisher's exact probability test.
Reproductive indices:
For each mating the following data were presented for each group: number of females placed with males, number of males mated with females, number of succesful copulation (= number of females mated), number of males that became sire, number of pregnant females as demonstrated by the presence of implantation sites observed at necropsy, number of females surviving delivery, number of females with liveborn and (all) stillborn pups, number of pups delivered (live- and stillborn), number of live pups at day n, number of pups lost, number of litters lost entirely, number of male pups at day n, number of implantation sites, number of lost implantations, litter size.

The following parameters were calculated:
- pre-coital time = time between the start of mating and successful copuls
- duration of gestation = time between gestation day 0 and day of delivery
- mating index = (number of females mated/number of females placed with males) x 100
- male fertility index = (number of males that became sire/number of males placed with females) x 100
- female fertility index = (number of pregnant females/number of females placed with males) x 100
- female fecundity index = (number of pregnant females/number of females mated) x 100
- gestation index = (number of females with live pups/number of females pregnant) x 100
- live birth index = (number of pups bom alive/number of pups born) x 100
- pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
- sex ratio day n = (number of live male pups on day n/ number of live pups on day n) x 100
- number of lost implantations = number of implantations sites - number of pups born alive
- post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
non-adverse effects only
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortalities occurred. One female of group E (257) had a complete degeneration of the right eye during pregnancy and lactation. This observation was not seen during premating and had no effect on the general behaviour of this rat. All other clinical signs listed are normal for rats of this strain and age.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Premating and mating period:
Occasionally statistically significantly effects were observed in the body weight change of males and females. These effects however, were inconsistent and not dose-related. In general no effect was observed on the mean body weight of males and females during the premating period. No effect was seen on the mean body weigth of the males during mating. The decrease in body weight change observed in females of all groups during premating days 86 to 93 is considered to be related to blood sampling for possible hormone analysis.

Gestation period:
The mean body weight of the female animals of groups C and D was statistically significantly increased as compared to the control group at gestation day (GD) 21. As a result the mean body weight change of females of group C was statistically significantly increased between GD 14-21. Since no effect was seen in the other groups, the effect on body weight in groups C and D is not considered to be related to the treatment.

Lactation period:
The mean body weight of the female animals of groups C and E was statistically significantly increased at postnatal days (PND) 1 and 4. As a result the mean body weight change was statistically significantly decreased in these groups during PND 4-7. At PND 21 the mean body weight in groups D and E was statistically significantly increased. Since no dose-relationship was observed these effects are not considered to be related to the treatmen.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Premating period:
Occasionally statistically significantly effects were observed in the food consumption expressed as g/animal and g/kg bw/d. No treatment related effects were seen.

Gestation period:
The mean food consumption, expressed as g/animal, of pregnant females was statistically significantly increased in groups C and F during GD 7-14. No effect was seen on the food consumption expressed as g/kg/day. No treatment related effects were seen.

Lactation period:
The mean food consumption was statistically significantly decreased in group F between PND 14-21 (g/animal/day and g/kg/day). The mean food consumption expressed as g/kg/day was statistically significantly decreased in groups E (PND 14-21) and group F (PND 7-14). This is considered to be treatment-related.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The number of estrus cycles in group F was statistically significantly increased as compared to the control group (3.8 versus 3.5 in the control group). The length of the longest cycle however, was not affected. The number of acyclic females was increased in the group F (17.9% versus 7.1% in the control group; not statistically significant) and the number of cyclic animals with a prolonged estrus period (> 1 day) was comparable in both groups. In conclusion, no adverse effects on estrus cyclicity were observed.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- Sperm motility: Sperm motility parameters were not affected.
- Epididymal sperm count: The number of spermatozoa per ml and number of spermatozoa per gram epididymal tissue was decreased in group F as compared to the control group (not statistically significantly). Since no clear dose-respons effect was seen this is not considered to be treatment related.
- Sperm morphology: Sperm morphology was not affected.
- Testicular sperm count: Testicular sperm count was not affected.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
In each group 28 females were placed with males after a premating period of 14 weeks. The number of females mated was 28,28,27, 28,28 and 28 in groups A, B, C, D, E and F, respectively. The precoital time in groups B and D was slightly increased. This was related to a higher number of females mated during the period 5-14 days from caging together as compared to the control group. The number of pregnant females was 26,27, 26, 26, 25 and 24 in groups A, B, C, D, E and F, respectively. All pregnant females had live pups except female B59. This female was mated, but did not deliver. At necropsy (32 days after mating) 5 implantation sites were present. The duration of gestation was comparable in all groups. Stillborn pups were present in groups C, D, E and F in 3.8; 3.8; 4.0 and 4.2 % of the pregnant females.
In conclusion no treament related effects were seen on fertility and reproductive performance.

The mean number of pups delivered was comparable in all groups. Pup mortality between PND 1-4 was 4.2; 2.7; 4.0; 1.8; 8.1 and 6.5% in groups A, B, C, D, E and F, respectively. Female E265 lost her total litter between PND 1-4. After PND 4 pup mortality was observed in group C only at a low incidence. The post implantation loss was 5.67; 11.54; 10.62; 4.83; 6.19 and 10.41% in groups A, B, C, D, E and F, respectively. The sex ratio was comparable in all groups. Mean pup weights at PND 1, 4, 7, 14 and 21 were comparable in all groups. In conclusion no treatment related effects were seen.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Males:
Mean data were calculated for 10 males per group. Absolute spleen weight in males of group F was statistically significantly increased as compared to the control group. Relative spleen weights were not affected. Relative brain weights in groups D and F were decreased. This however, is correlated to the increased terminal body weight in these groups.

Females:
Absolute weight of the kidneys in group D and group F was statistically significantly increased as compared to the control group; relative weight was comparable to the control group. Relative brain weight in group C, D and E was statistically significantly decreased; this was most probably related to the increased terminal body weight. Relative liver weight of the animals of group E and F was statistically significantly decreased when compared to the control group. The relative difference as compared to the control group was up to 7% and not related to changes in terminal body weight. Since no effect on liver was observed at microscopic examinations and biochemistry results of the 13-week toxicity study, this finding is not considered to be treatment related.

In conclusion no treatment related effects were seen on organ weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At necropsy, no treatment related gross changes were observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopical examination did not reveal treatment related histopathological changes. The histopathological changes observed were considered unrelated to treatment because they were about equally distributed amongst the different groups or occurred in a single or a few animals only. Therefore, the differences in incidence were considered fortuitous findings.

Non-mated female CI65 showed no histopathological changes in the reproductive organs. Female CI65 was caged together with male CI66. At autopsy this male showed a strongly decreased testes and epididymides weight. All other organ weights were comparable with control values. Male CI66 had no motile sperm and the epididymal sperm count was zero. The testicular count, however, was lower than the control values but sperm cells were present.

The following females were non-pregnant and the relevant histopathological changes observed in the reproductive organs are given between brackets: A5 (ovarian cyst(s)), B105, C131, D177 (moderate mononuclear cell infiltrate vagina), D221, E235, E253 (ovarian cyst(s)), E271, F319 (pituitary cyst(s)), F321, F327 (pyometra, endometritis, vaginitis) and F335 (ovarian cyst(s)). Ovarian cyst(s) may cause hormonal unbalance which can prevent a female from gestation. However, there were also females with ovarian cyst(s) that became pregnant. The endometritis and pyometra in F327 most likely prevented gestation. The influence of the vaginitis in D177 on gestation is uncertain. All other non-pregnant females did not show histopathological changes in their reproductive organs which could explain the failure to become pregnant. No effects on organ weights were observed.

The following males had failed to sire: A6, A18, B106, C132, C166, D178, D222, E236, E254, E272, F320, F322, F328 and F336. Male B60 mated succesfully, but the female became 'pregnant with implantations but no pups'.
Observations of CI 66 are already described above. Male F322 had no motile sperm. At autopsy this male showed a stronely decreased testes and epididymides weight. The epididymal sperm count was zero. The testicular count, however, was lower than the control values, but sperm cells were present. Microscopical examination revealed that F322 had testes with very severe atrophy of the seminiferous tubules and epididymides devoid of spermatozoa, which most probably had caused the animal failing to sire. Male F290 showed a strongly decreased testes and epididymides weight. All other males that had failed to sire did not show histopathological lesions in their reproductive organs which could explain their failing to sire.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
3 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Actual received dose based on 5% w/w L-isoleucine diet.
Dose descriptor:
NOAEL
Effect level:
2 720 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Remarks on result:
other: No adverse effects at highest tested dose.
Dose descriptor:
NOAEL
Effect level:
4 280 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Remarks on result:
other: No adverse effects at highest tested dose.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
inccreased number of runts
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

The mean number of pups delivered was comparable in all groups. Pup mortality between PND 1-4 was 4.2; 2.7; 4.0; 1.8; 8.1 and 6.5% in groups A, B, C, D, E and F, respectively. Female E265 lost her total litter between PND 1-4. After PND 4 pup mortality was observed in group C only at a low incidence. The post implantation loss was 5.67; 11.54; 10.62; 4.83; 6.19 and 10.41% in groups A, B, C, D, E and F, respectively. The sex ratio was comparable in all groups. Mean pup weights at PND 1, 4, 7, 14 and 21 were comparable in all groups. In conclusion no treatment related effects were seen.

A statistically significantly increase was seen in the number of runts in group F at PND 21. In group E the number of runts was also increased, but at a lower litter incidence (litter incidences were 3, 1, 3, 1,2 and 6 for groups A, B, C, D, E and F, respectively). Other clinical observations were seen at low incidences in pups among all groups.
During autopsy at PND 21 observations were: small brains, pink appearance of the thymus, hydrogephrosis, small kidney, flabby kidney, pale appearance of the kidney, agenesis of the anus, overfilled rectum and red spots on the thymus. The observations were divided among the groups with the highest pup and litter incidence in the control group.
In stillborn pups and pups that died during PND 1-21 no abnormalities were observed during macroscopic examinations.

The statistically significantly increased number of runts at PND 21 in group F is considered to be treatment related.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
3 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Actual received dose based on 5% w/w L-isoleucine diet.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Test substance intake (g L-isoleucine/kg bw weight/d)

Group

Dietary level

(% w/w)

Mean intake males

(premating – necropsy)

Mean intake females

(premating – gestation – lactation)

A

0

0

0

B

1 %

0.55

0.88

C

2 %

1.08

1.75

D

3 %

1.58

2.61

E

4 %

2.11

3.43

F

5 %

2.72

4.28

 

Applicant's summary and conclusion

Conclusions:
5% w/w L-isoleucine (approx. 3.5 g/kg bw/d) is considered to be the NOAEL for parental toxicity, toxicity to the offspring and reproductive toxicity.
Executive summary:

The objective of this one-generation reproductive toxicity study was to provide data on the possible effects of L-isoleucine on the estrus cycle, the male and female reproductive performance, and the growth and development of the offspring. The study comprised the following groups: control (A); 1% w/w L-isoleucine (B); 2% w/w L-isoleucine (C); 3% w/w L-isoleucine (D); 4% w/w L-isoleucine (E) and 5% w/w L-isoleucine (F). Twenty-eight Wistar rats, Crl:WI(WU) per sex were allocated to each group and rats were fed with diets containing the active ingredient L-isoleucine.

Measurements and observations during the study comprised: analysis of content in diets, clinical signs, body weight, food consumption, estrus cycle, fertility, parturition, litter development, sperm analysis, gross necropsy, organ weights and microscopic evaluation.

The content of the test substance L-isoleucine in all diets measured was close to the intended concentrations. No mortalities occurred and no effect was seen on clinical signs.

No effect was observed on body weight during premating, gestation and lactation. The mean food consumption expressed as g/kg/day was statistically significantly decreased in group E on postnatal day (PND) 14-21 and in group F during PND 7-14.

No adverse effect on estrus cyclicity was observed and sperm analysis parameters were not affected. No treatment related effects were seen on fertility, reproductive performance, litter size, sex or pup weight.

The number of runts was statistically significantly increased at PND 21 in group F.

At necropsy no treatment related gross changes were observed. Microscopical examination did not reveal treatment related histopathological changes. No effects was seen on parental and pup organ weights.

Histopathological examinations of reproductive organs in non-mated and non-pregnant females and males that did not sire did not reveal treatment-related effects.

In conclusion statistically significant effects were seen on maternal food consumption during lactation at dose levels of 4 and 5% w/w L-isoleucine and on the number of runts at the end of the lactation period at a dose level of 5% w/w L-isoleucine. Since these findings were not reflected in the mean body weight of females or mean pup weights, the effects are not considered to be adverse. Based on the results of this study, 5% w/w L-isoleucine (corresponding to approximately 2.72 g/kg body weight/day in males and 4.28 g/kg body weight/day in females) is considered to be the No Observed Adverse Effect Level (NOAEL) for parental toxicity, toxicity to the offspring and reproductive toxicity.