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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: According to GLP and comparable to guideline study with restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: "Toxicity Test Guideline for Pharmaceutical Products", Ministry of Health and Welfare, Yakushin 1 No. 24, 11 September 1989.
Deviations:
not specified
Remarks:
This guideline was not available. Compliance with OECD 414 was checked instead.
Qualifier:
according to guideline
Guideline:
other: "Revision of Test Guideline for Pharmaceutical Products in relation with Fertile-developmental Toxicity", Pharmaceutical Affairs Bureau Examination Section Manager, Ministry of Health and Welfare, Yakushin No. 316, 4 April 1997.
Deviations:
not specified
Remarks:
This guideline was not available. Compliance with OECD 414 was checked instead.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See below.
Principles of method if other than guideline:
The study design agreed with OECD 414 (2001), with the following deviations. The scheme for dose administration (day 7 through 17 of presumed gestation) does not agree with OECD 414 (2001), which states day 5 post mating to the day prior to scheduled caesarian section. The examination of foetal ossification was performed on a limited number of bones.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
L-lysine hydrochloride
EC Number:
233-234-9
EC Name:
L-lysine hydrochloride
Cas Number:
10098-89-2
IUPAC Name:
L-lysine hydrochloride
Details on test material:
- Name of test material (as cited in study report): L-lysine hydrochloride
- Substance type: white powder
- Physical state: solid
- Analytical purity: 99.6%
- Lot/batch No.: 0045081603
- Storage condition of test material: Ambient temperature (19-25˚C)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Japan Inc.
- Age at study initiation: 11-12 weeks (F) and 12-13 weeks (M)
- Weight at study initiation: On the date of receipt, body weight ranged from 358 to 387 g for male rats and from 216 to 256 g for female rats. Mean weight per test group at the start of dose administration: 265.2-265.4 g (SD 8.3-10.0 g)
- Fasting period before study: none
- Housing: individually housed in stainless steel wire-mesh cages except during mating
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-25°C).
- Humidity (%): 36-63%
- Air changes (per hr): 10-15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark
IN-LIFE DATES: From: March 27, 2006 To: April 24, 2006

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
water for injection
Details on exposure:
Dose levels were 0, 300 and 1000 mg/kg bw/day, administered from day 7 through 17 of presumed gestation (female rats with a copulation plug or the presence of spermatozoa in a vaginal smear were considered to be at day 0 of presumed gestation).
VEHICLE
- Justification for use and choice of vehicle (if other than water): not relevant
- Concentration in vehicle: 3% and 10% w/v.
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): 5184, 5E78
- Purity: Japanese Pharmacopeia
- Other: Test solutions were prepared at the time of use and used within 3 hours of preparation.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: one night
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: no data
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
From day 7 through 17 of presumed gestation (female rats with a copulation plug or the presence of spermatozoa in a vaginal smear were considered to be at day 0 of presumed gestation).
Frequency of treatment:
Once daily
Duration of test:
20 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
20 pregnant females (20 females had copulated, resulting in the 20 pregnant females on test)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high dose level of 1000 mg/kg was selected since it it is the maximum administrable dose level for rats exceeding the expected clinical use dose level; the low dose level of 300 mg/kg was selected as a level of one third of the high dose level.
- Rationale for animal assignment (if not random): Mated females were allocated to the study, ensuring that animal body weights were evenly distributed amongst groups on the day of confirmed mating (Day 0 of gestation), using Block Allocation method with a computer program.
- Other:

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed at least 3 times (pre-dose, post-dose and 2hr after dose) daily during the administration period. For remaining period, animals were observed once a day (morning time) for any abnormalities in general conditions including appearance, excreta, nutritional condition, posture and behavior.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, 4 through 18 and day 20
FOOD CONSUMPTION: Yes
- Time schedule for examinations: on day 1, 4, 8, 11, 14, 18 and 20
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: macroscopic examination of tissues and primary organs in thoracic and abdominal cavities.
OTHER:
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: no (instead the placenta was weighed)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: numbers of live and dead fetuses were recorded.
Fetal examinations:
- External examinations: Yes: all per litter; fetuses were also weighed and sex was determined
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter; examination for skeletal malformations and variants; to evaluate the progress of the ossification, the number of each ossification of metacarpal bones, metatarsal bones and sacral vertebrae as well as a sternum nucleus ossification rate were examined.
- Head examinations: No
Statistics:
Means and standard deviations of body weight, body weight gain (Day 7 thru 18 and Day 18 thru 20 of gestation), food consumption, number of corpora lutea, number of implantation, and number of live fetuses were calculated for each of test group. Litter mean fetal weight was calculated and group means and standard deviations also calculated for each of test group. These means and standard deviations were tested for equal distribution using Bartlett's test with significance level of 1% on two tailed analysis. As homogeneity was noted, Dunnett's test was applied with significance level of 5 and 1 % on two tailed analysis. Litter means and standard deviations were calculated for metacarpal bone, metatarsal bone, sacral vertebrae and sternum nucleus ossification rate, of which the group mean and standard deviations were also calculated and were subjected to the Student's t test (significant level of 5 and 1 % on two tailed analysis) for analysis of difference after confirmation of homogeneity using F test (significance level of 5 % on two tailed analysis).
Litter means and standard deviations of implantation index, embryo/fetuses mortality, external abnormality index, visceral abnormality index, visceral variant index, skeletal abnormality index, skeletal variant index and sternum nucleus ossification rate were calculated for each test group and statistically analyzed with Steel's method (No. of group = 3, significant level of 5 and 1 % on two tailed analysis) and the rank sum test of Wilcoxon (No. of group = 2, significant level of 5 and 1 % on two tailed analysis). As to the sex ratio, total numbers of live male and fetuses for each test group were analyzed using Chi square analysis with Yate's continuity correction (significant level of 5 and 1 % on two tailed analysis).
Historical control data:
No data. However the report referred to background data in its discussion: "... the changes and variants noted in the visceral and skeletal examination were thought to be of natural causes and fall in the range of historical background data of this laboratories".

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
There were neither deaths nor clinical abnormalities in the control and treated groups. There were no effects on body weight, body weight gain and food consumption. Gross necropsy revealed no abnormalities.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The number of corpora lutea, number of implantations, embryo / fetal mortality, the number of live fetuses, fetal sex ratio and fetal body weight were comparable in treated groups and controls. There were no fetuses with external abnormalities in the test and control groups and no abnormalities were observed in the placenta of live fetuses.
In the control group there were 8 live fetuses with visceral abnormalities in 7 litters, while there were 9 live fetuses in 6 litters in the group treated with 1000 mg/kg. Live fetuses with visceral variants were found total 6 in 5 litters in control and total 8 fetuses in 6 litters in the group treated with 1000 mg/kg. There were no live fetuses with skeletal abnormalities. Live fetuses with skeletal variants were noted in 6 litters for 10 fetuses in the control group, and for total 21 fetuses in 9 litter of the group treated with 1000 mg/kg. The incidences of these abnormalities and variants were not remarkably different between the 1000 mg/kg bw/day group and the control, and they were not statistically significant. As to the progress of ossification, there were no significant differences in the number of metacarpal bone, metatarsal bone, sacral vertebrae and sternum nucleus ossification rate between the 1000 mg/kg bw/day group and the control group.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Maternal and developmental NOAEL ≥1000 mg/kg bw/day; study agreed with OECD 414 (2001), except the treatment schedule (day 7 through 17 instead of day 5 post mating to the day prior to scheduled caesarian section) and limited number of bones examined for foetal ossification.

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