Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-04-21 to 2005-12-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
Annex 2F to Commission Directive 2004/73/EC, Official Journal of the European Communities, Ll52, 30.4.2004
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Wistar outbred (Crl:(WI)WU BR)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: females ca. 11 weeks, males ca. 12 weeks
- Weight at study initiation: 197.4 g as a mean (179 g - 220.6 g) of females at day 0 of gestation
- Fasting period before study: none
- Housing: Males individually in macrolon cages type 3 (42.5x26.6x15 cm), females before mating in groups of 4 in macrolon cages type 4 (48x37.5x21cm) with sterilized wood shavings (Lignocel, Type 3/4) as bedding material and shreds of paper (Enviro-dri) as environmental enrichment. For mating 2 females were placed together with 1 male in macrolon cages type 3. Mated females were housed individually in the same type of cages which were placed in another cage rack.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
According to guideline

IN-LIFE DATES: From: 2005-04-27 To: 2005-05-30 - 2005-06-2, depending on day of pregnancy. Non-mated females were sacrificed after the mating period.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Not applicable as the test substance as such was mixed with the diet.

DIET PREPARATION
- Rate of preparation of diet (frequency): Diet mixed once with test substance shortly before the start of the study. The resulting experimental diet was stored.
- Mixing appropriate amounts with (Type of food): Rat&Mouse No. 3 Breeding Diet, RM3; powdered. From SDS Special Diets Services, Witham, England.
- Storage temperature of food: < - 18 °C.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
1. Analysis of L-valine in diet
1.1 Principle
The analysis of L-valine is based on a TNO Standard Operating Procedure for the analysis of free amino acids in feed. After addition of norvaline as internal standard, samples of rodent diet were extracted with an aqueous solution of trichloroacetic acid. The concentration of L-valine was determined using ion chromatography after post-column reaction with ninhydrin reagent (amino acid analyser). Quantitation was obtained by comparing the peak areas of L-valine and norvaline in the sample extracts with those of a calibration solution containing known amounts of L-valine and norvaline.
1.2 Validation criteria
The method should conform to the following criteria:
recovery: the recovery of the test substance from test diet should be between 80 and 110 % at each of the three concentration Ievels used in the toxicity study;
repeatability: the relative standard deviation in the percentage recovery found upon analysis of five spiked diet samples per concentration Ievel (the same three Ievels as used in the recovery test) should be less than 10 %.
1.3 Preparation of validation samples
Validation samples with nominal concentrations of 0.2 %, 1 % and 5 % L-valine, respectively, were prepared, in five-fold, by addition of 0.800 ml, 4.00 ml and 20.00 ml, respectively, of a L-valine stock solution, to 5.0 g (accurately weighed) rodent diet.
The validation samples and the blank diet (control) were analysed.
1.4 Sample treatment
To 5 g of diet (accurately weighed) a calculated amount of an internal standard solution (dl-norvaline) was added. Subsequently, a volume of trichloroacetic acid (TCA) solution was added to the diet samp1e and the mixture was stirred for approximately 1 hour and left to settle for 15 minutes. The extract was diluted with Iithium citrate buffer solution (0.1 mol/l, pH 2.2), filtered (0.45 µm) and analysed.
1.5 Chromatography
The followving LC conditions were used:
Analyser : JEOL AminoTac JLC-500/V amino acid analyser
Column : 4 x 120 mm, tandem packed column
Column temp. : gradient
Injection volume : 50 µl
Detection : 570 nm after post-column reaction with ninhydrin reagent
Reaction temp. : 135 °C
pH of lithium citrate buffer solutions for JEOL AminoTac JLC-500/V amino acid analyser (obtained
from JEOL):
Lithium citrate buffer solution pH
1st buffer 2.98
2nd buffer 3.28
1.6 Calibration
For calculation of the amount of L-valine in the test substance "L-valine" and in the validation samples, a calibration solution was prepared containing 21.40 mg valine and 25.81 mg norvaline per Iiter Iithium citrate buffer solution (0.1 mol/1, pH 2.2) as follows: 200 µl of a stock solution containing 0.1070 g dl-valine (Sigma > 97 %) /50 ml 0.1 mol/1 HCI and 20 µI of a stock solution containing 2.5807 g dl-norvaline (Fluka, PA) /100 ml 0.1 mol/1 HCl, were
added to a 20 ml volumetric flask, which was brought to volume with Iithium citrate buffer solution (0.1 mol/1, pH 2.2).
For calculation of the amount of the test substance L-valine in all diet samples, a calibration solution was prepared containing 20.92 mg of the test substance L-valine and 24.85 mg norvaline per Iiter Iithium citrate buffer solution (0.1 mol/1, pH 2.2) as follows: 200 µl of a stock solution containing 0.1046 g test substance L-valine / 50 ml 0.1 mol/l HCl and 20 µl of a stock solution containing 2.4851 g dl-norvaline (Fluka, PA) / 100 ml 0.1 mol/l HCl, were added to a 20 ml volumetric flask, which was brought to volume with Iithium citrate buffer solution (0.1 mol/1, pH 2.2). The calibration solution was stored at 2- 10 °C.


2. Determination of homogeneity, stability and content of L-valine diet
2.1 Homogeneity
The homogeneity of diets containing the test substance L-valine was determined in the first batch of diets prepared for studies on 2 May 2005. Five samples per dose level, taken at different locations in the feed container, were analysed to determine homogeneity. For each dose level, a one way analysis of variance (Anova) was performed using place as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly at the five locations in the feed container). The test substance L-valine was considered tobe homogeneously distributed in the diet if p > 0.01 and/or if the relative standard deviation (RSD) between the mean concentrations at the five locations is less than 5%.

2.2 Stability
The stability of the test substance L-valine in diet under simulated experimental conditions was determined by analysis of the first batch of diets prepared for the studies on 2 May 2005, after storage at room temperature in an open container for 7 days and after storage at < -18 °C in a closed container for 5 weeks. For each dose level, a one way analysis of variance (Anova) was performed using time as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the difference between the results of the first day (t = 0) and the day on which the analysis is repeated, is significant). If p ~ 0.01 and/or if the relative decrease in the mean concentration on storage is smaller than 10 %, the test substance L-valine was considered to be stable in the diet over that period.

2.3 Content
The content of the test substance L-valine was determined in diets prepared on 2 May 2005, 2 June 2005 and 1 July 2005. The content of the test substance L-valine in diet was considered to be 'close to intended' if the difference between the mean concentration and the intended concentration is less than 10 %.


2.4 Determination of valine in the test substance L-valine
The amount of the active ingredient valine in the test substance L-valine (g/ 100 g) was determined in triplicate, using essentially the same method as described for the diet samples: To 0.2 g test substance (accurately weighed) 8.00 ml internal standard solution was added. Subsequently, 8 ml trichloroacetic acid solution (20 %) and 34 ml trichloroacetic acid solution (10 %) were added to the sample and the mixture was stirred for approximately 1 hour. 50 µl of this solution was diluted with 10 ml Iithium citrate buffer solution (0.1 mol/1, pH 2.2), filtered (0.45 µm) and analysed as described above.

3. Results
4.1 Validation of the analytical method for the determination of the test substance L-valine in diet
The recoveries measured for L-valine in diet were 100 %, 104 % and 101 %. respectively. These values met the criterion set for recovery (recovery between 80 % and 110 %). The relative standard deviation (RSD) in the percentage recovery of L-valine for each validation Ievel was 1.1 %, 1.2 % and 1.2 %, respectively. These values are well below the criterion set for the RSD in the percentage recovery (RSD < 10 %).
3.2 Homogeneity, stability and content of L-valine in diet
3.2.1 Homogeneity
Since the criteria for homogeneity were met for all dose Ievels (relative standard deviation (RSD) between the mean concentrations at the five different locations is less than 5 % and/or F-probability > 0.01), the test substance L-valine was considered to be homogeneously distributed in the diets.
3.2.2 Stability
The relative differences measured after storage at room temperature for 7 days and after storage at <-18 °C for 5 weeks, were less than 10 % for all dose Ievels. Therefore, the test substance L-valine was considered stable in rodent diet at all dose Ievels upon storage at room temperature for 7 days and upon storage at < -18 °C for 5 weeks.
3.2.3 Content
The concentrations were 'close to intended' (relative difference less than 10 %) for all dose Ievels of the test substance in diet, except for the 1 % dose Ievel prepared on 2 May 2005, for which the criterion for content was just exceeded (relative difference was +12 % ).
3.3 L-valine in the test substance "L-valine"
The mean measured amount of L-Valine in the test substance "L-valine" was 98.0 g / 100 g.

4. Conclusions
The method used for the quantitative analysis of the test substance L-valine in rodent diet met the validation criteria.
The test substance L-valine was homogeneously distributed in the diets prepared.
The test substance L-valine was considered to be stable in rodent diet when stored at room temperature for 7 days and when stored at < -18 °C for 5 weeks.
The content of the test substance L-valine in all diets prepared was close to the intended concentrations, except for the 1 % dose level, for which the criterion for content was just exceeded (relative difference was +12 %).
The mean measured amount of L-valine in the test substance "L-valine" was 98.0 g/100 g.



Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1: 2
- Length of cohabitation: Overnight to maximum of 4 days
- Further matings after two unsuccessful attempts: Females were left until mating. Non-mated females were excluded from the further study.
- Verification of same strain and source of both sexes: yes - by the supplier
- Proof of pregnancy: sperm in vaginal smear referred to as GD 0 of pregnancy
Duration of treatment / exposure:
The test substance was administered via the diet throughout the entire study.
Frequency of treatment:
Continouous throughout the study.
Duration of test:
21 days after gestation.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 % L-valine: Intake 0 g/kg bw / day (control)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.2 % L-valine: Intake 0.10 - 0.15 g/kg bw / day (mean: 0.13 g/kg bw / day)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1 % L-valine: Intake 0.46 - 0.75 g/kg bw / day (mean: 0.61 g/kg bw / day)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
5 % L-valine: Intake 2.09 - 3.46 g/kg bw / day (mean: 2.76 g/kg bw / day)
Basis:
nominal in diet
No. of animals per sex per dose:
28
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Intended use of the test substance in animal feed and the potentially resulting oral exposure of humans
- other: The oral route was used because this is an anticipated route of exposure of animals and humans (through the consumption of animal products as food).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each female daily in the morning
- Cage side observations checked in table were included: Not in table, summary of results given

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Each mated female on GD 0, 3, 7, 10, 14, 17 and 21

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: Uteri, ovaries, placentas

OTHER:
- Fetopathological examination: 50 % of the fetuses examined for soft tisse anomalies. 50 % of the fetuses examined for skeletal abnormalities. Skeletal and visceral examinations on all fetuses.
- Fertility and reproductive performance: Female fecundity index, pre-implantation loss, post-implantation loss, gestation index and sex ratio were calculated for each dose group.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
- number of live and dead fetuses
- sex of the fetuses
- number of grossly visible malformed fetuses and fetuses with external abnormalities
- weight of ovaries
- weight of uterus, containing placentas and fetuses
- weight of uterus, empty
- weight of fetuses
- weight of the placentas
- gross evaluation of the placentas
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [all per litter for general condition / half per litter for abnormalities after staining]
- Head examinations: Yes: As to skeleton.
Statistics:
The resulting data were analysed using the methods mentioned below.
- 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 tests.
- Fisher's exact probability test was used to evaluate the number of mated and pregnant females and females with live fetuses.
Number of corpora lutea, pre- and post-implantation loss, live and dead fetuses and early and Iate resorptions were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U-test. Fisher's exact probability test was used to evaluate the data of the fetopathological screening.
- Statistical evaluations on variables associated with the fetuses were considered on a litter basis in accordance to standard procedures. Additional evaluations on a fetus basis were perforrned to attempt to identify any specific dose-related effect that may have occurred. As a Ievel of significance was considered: p < 0.05
Indices:
- Female fecundity index = (number of pregnant females I number of females mated) x 100
- Pre-implantation loss = [(number of corpora lutea - number of implantation sites) / number of corpora lutea] x 100
- Post-implantation loss = [(number of implantation sites - number of live fetuses) / number of implantation sites] x 100
- Gestation index = (number of females with live fetuses I number of females pregnant) x 100
- Sex ratio = (number of live male fetuses I number of live fetuses) x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
In the 5% L-valine group ( equivalent to 2. 76 g/kg body weight/day) treatment-related effects were observed on food-consumption, body weight change, carcass weight (terminal body weight minus gravid uterus weight), and net weight change (carcass weight minus body weight day 0).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
610 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
Equivalent to 1 % L-valine in diet
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
In the 5% L-valine group ( equivalent to 2. 76 g/kg body weight/day) treatment-related effects were observed on fetal weights.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
610 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
Equivalent to 1% L-Valine in diet
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
changes in litter size and weights
external malformations
skeletal malformations
other: grossly visible abnormalities; soft tissue abnormalities;

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Feeding of L-valine at dietary Ievels up to 1% ( equivalent to 0.61 g/kg body weight/day) was tolerated without obvious signs of maternal and developmental toxicity. NOAEL = 610 mg/kg bw/day.