Lysine hydrochloride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Reference 1

Endpoint:

fertility, other

Type of information:

(Q)SAR

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE:QSAR Toolbox 4.4; Database version: 4.4

2. MODEL (incl. version number): Androgen Receptor Antagonism (Human in vitro) - Danish QSAR DB battery model (v.1.0)

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: Cl.NCCCCC(N)C(O)=O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: Androgen Receptor Binding - Human Health Hazards -> Toxicity to
Reproduction -> AR antagonism -> Homo sapiens
- Defined domain of applicability: The target chemical FALLS within the applicability domain.

Qualifier:

no guideline available

Principles of method if other than guideline:

SAR/QSAR prediction
prediction based on Androgen Receptor Antagonism (Human in vitro) - Danish QSAR DB battery
model

GLP compliance:

no

Dose descriptor:

other: Androgen Receptor Binding

Effect level:

other: negative

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: roles in male reproductive development and function and in maintaining female fertility through key roles in the regulation of follicle health, development, and ovulation

Remarks on result:

other: The QSAR calculation showed a negative results for reproductive toxicity based on androgen receptor binding. An effect level is not necessary due to high intake through food of this essential nutrient.

Dose descriptor:

other: Androgen Receptor Binding

Generation:

F1

Effect level:

other: fffnegative

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: roles in male reproductive development and function and in maintaining female fertility through key roles in the regulation of follicle health, development and ovulation

Remarks on result:

other: The QSAR calculation showed a negative results for reproductive toxicity based on androgen receptor binding. An effect level is not necessary due to high intake through food of this essential nutrient.

Reproductive effects observed:

no

Conclusions:

Based on the QSAR model Androgen Receptor Binding as the endpoint for reproductive toxicity showed a negative result for Lysine-HCl.

Executive summary:

The Androgen Receptor Antagonism model is suitable for the prediction of reproductive toxicity. The androgen receptor (AR) has defining roles in male reproductive development and function as well as in regulating follicle development and ovulation (Walters K.A., Allan C.M., Handelsman D.J., 2008, BIOLOGY OF REPRODUCTION 78, 380–389).

Therefore, this QSAR prediction together with all available data showed no evidence of reproductive toxicity of L-lysine-HCl.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Scientific study not performed according to common guidelines. The feeding conditions: fed with lysine-excess diet in this study. Dosing of females from day 1-21 of pregnancy.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Scientific study in which the effects of dietary excess of lysine on maintenance of pregnancy and reproductive performance, including fetal growth in rats was examined.

GLP compliance:

not specified

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Route of administration:

oral: feed

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

from day 1 to day 14 or 21 of pregnancy

Dose / conc.:

5 other: %

Remarks:

nominal in diet

No. of animals per sex per dose:

5 rats in the 5% Lysine group
7 rats in the ad libitum control group
5 rats in the pair-fed control

Control animals:

yes

Details on study design:

Two experiments were carried out:
Experiment 1: the effect of amino acid-excess diets on maintenance of pregnancy was examined. For this purpose pregnant animals receiving the experimental diets were killed on day 14 of pregnancy and the products of conception (uterus, amniotic fluid, placenta, and fetuses) were weighed, because nutritionally induced fetal resorption begins on about day 10 of pregnancy.
Experiment 2: the effects of excess of lysine on the nitrogen balance of the dams, the free amino acid concentrations in maternal plasma and fetal brain and fetal growth were examined.

Parental animals: Observations and examinations:

BODY WEIGHT: Yes, daily
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, daily

Litter observations:

Fetuses weights,
Litter size

Reproductive indices:

Weight of products of conception,
placenta weight,
fetuses weights,
fetal resorptions

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

During pregnancy, daily food intakes initially increased gradually, but near term they decreased appreciably in all pregnant rats.

Reproductive performance:

no effects observed

Description (incidence and severity):

Pregnancy was maintained in 100% of the rats on diets with an excess of lysine.
The weights of conception products were similar to those of the corresponding pair-fed control.
But given excess lysine, the weights of fetuses were significantly lower than those of the control.
The litter size was normal in all.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Given excess lysine, the weights of fetuses were significantly lower than those of the control.

Other effects:

no effects observed

Description (incidence and severity):

The fetal brain concentration of given excess lysine the brain con­centration of this amino acid was comparable to those of the control.
Ingestion of diet containing excess lysine did not result in appreciable alterations in plasma or brain amino acid concentrations.

Conclusions:

This study on effects of dietary excess of lysine on maintenance of pregnancy and reproductive performance, including fetal growth in rats showed that pregnancy was maintained in 100% of the rats on diets with the excess of lysine; the weights of conception products were similar to those of the corresponding pair-fed control; the weights of fetuses were significantly lower than those of the control; the litter size was normal in all; did not result in appreciab]e alterations in plasma or brain amino acid concentrations.

Executive summary:

The effects of diet with an excess of lysine on the maintenance of pregnancy and fetal growth in rat were investigated in this study. Female Sprague Dawley rats weighing about 190 g were fed on 6 % casein diet (a low protein diet) containing 5% Lysine from day 1 to 14 or 21 of pregnancy (also the day of autopsy). Pregnancy was maintained in 100 % of the rats on diets with an excess of lysine. During pregnancy, daily food intakes initially increased gradually but near term they decreased appreciably in all pregnant rats. With regard to lysine in excess, the body weight gain during pregnancy for the groups receiving lysine were almost comparable to that of the pair-fed control. In all animals given excess amino acid lysine the weights of conception products were similar to those of the corresponding pair-fed controls. However, in most groups given excess amino acid lysine, the weight of foetuses was significantly lower than those of the respective pair-fed controls. The average weight of the foetuses of the groups receiving 5% excess lysine was 3.12 ± 0.30 g compared to 4.11 ± 0.22 g for their pair-fed control. The litter size was almost normal in all rats on excess amino acid diets compared to the controls. The concentrations of lysine in the maternal plasma were not increased appreciably by excess amounts of lysine in the diet. Ingestion of diet containing excess lysine did not result in appreciable alterations in plasma or brain amino acid concentrations.

Additional information

Short description of key information:

The study of Matsueda and Niijama (1982) on effects of dietary excess of lysine on maintenance of pregnancy and reproductive performance, including fetal growth in rats showed that pregnancy was maintained in 100% of the rats on diets with the excess of lysine; the weights of conception products were similar to those of the corresponding pair-fed control; the weights of fetuses were significantly lower than those of the control; the litter size was normal in all; did not result in appreciable alterations in plasma or brain amino acid concentrations.

The QSAR model Androgen Receptor Binding as the endpoint for reproductive toxicity showed a negative result for the prediction of reproductive toxicity of Lysine-HCl. The Androgen Receptor Antagonism model is suitable for the prediction of reproductive toxicity. The androgen receptor (AR) has defining roles in male reproductive development and function as well as in regulating follicle development and ovulation (Walters K.A., Allan C.M., Handelsman D.J., 2008, BIOLOGY OF REPRODUCTION 78, 380–389).

L-Lysine HCl showed no effects on the reproductive organs in general repeated dose and developmental toxicity studies and the QSAR prediction on reproductive toxicity. Moreover, lysine is an essential amino acid that is required for normal functioning of humans.

Therefore, along with all available data there is sufficient weight of evidence for no concern of reproductive toxicity of L-lysine-HCl and no further test is required for this endpoint.

Effects on developmental toxicity

Description of key information

A pre-natal study was performed according to OECD 414 (2001), except for 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 were examined for foetal ossification. The NOAELs for maternal and developmental toxicity were set at ≥1000 mg/kg bw/day. L-lysine hydrochloride supplemented in excess in food may affect pegnancy as laid down in the conclusion of the second study.

The study of Funk et al. (1991) examined the effects of supplemental Lysine on pregnancy course with levels of Lysine at 50%, 100% and 500% excess over the levels of Lysine present in the control diet. There were no statistically significant differences within Lysine groups in the mean number of viable foetuses and in the mean number of early resorptions. The total fetal weight in each litter did not differ significantly among the Lysine groups, their pair-fed controls, or the control group. L-500 had significantly smaller average fetal weights than all other Lysine groups. The rats receiving the highest amount of lysine (L-500) had foetuses that were significantly shorter than all the other Lysine groups with the exception of the pair-fed control LC50). No significant differences in the ratio of males to females were found within the Lysine groups. No obvious congenital malformations or abnormalities were observed in any of the foetuses. In conclusion, the results of this study suggest that variable levels of lysine supplementation do not cause grossly recognizable fetal malformations in rats. Lower supplementation levels of lysine do not appear to have any negative impact on maternal weight gain, fetal weight or other fetal parameters that may signify toxicity. Diets supplemented with a high amount of lysine (500% excess) appear to be associated with a lower than expected maternal weight gain and a significantly reduced fetal weight. The L-500 diet (500% excess lysine) relates to human supplementation with lysine of approximntely 35 g per day. These levels of amino acid supplementation are clearly higher than those reported in human trials. For example, the well-balanced and protein-adequatic North American diet contains about 5-8 g of lysine daily. Recent intake recommendations to meet the lysine requirement range from 64 to 30 mg/(kg / d) for 0.5-y infants and adults (>18 y), respectively. Lysine intake in the Western human diet is in the range 40–180 mg/(kg/d). An upper limit of 300–400 mg/kg/d can be considered in humans (Tsubuku et al, 2004). (Tomé D, Bos C, 2007, The Journal of Nutrition. 137 (6 Suppl 2): 1642–1645). This corresponds to a maximum of 28 g/day Lysine for an adult weighing 70 kg.

Link to relevant study records

Reference

Reference 1

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.

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

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

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

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:

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".

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.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

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.

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.

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not described

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication/study report which meets basic scientific principles.

Qualifier:

no guideline followed

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:

not applicable

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Tyler Laboratories (Bellevue, WA)
- Age at study initiation: not described
- Weight at study initiation: not described
- Fasting period before study: no
- Housing: individually in screen bottom cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): adlibitum
- Acclimation period: not described

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 20◦C
- Humidity (%): not described
- Air changes (per hr): not described
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: not described

Route of administration:

oral: feed

Type of inhalation exposure (if applicable):

not specified

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
Rats of the control group or the pair-fed control group were fed with modified American Institute of Nutrition AIN-76 semi-purified diet.
- Rate of preparation of diet (frequency): The powdered diet was mixed in large batches to minimize variation among groups
- Mixing appropriate amounts with (Type of food): After addition of oik, a part of the diet was separated as basis diet (control diet). L- lysine was added in predetermined amounts to the basic diet.
- Storage temperature of food: stored in plastic bags, labeled and frozen until use and refrigerated between feeding

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

not applicable

Details on mating procedure:

animals were time-mated by the breeder

Duration of treatment / exposure:

from day 0 until day 20 of pregnancy continuously via diet

Frequency of treatment:

from day 0 until day 20 of pregnancy

Duration of test:

20 days

Dose / conc.:

77 900 mg/kg diet

Remarks:

L-lysine at 500% excess over controls. Basis: nominal in diet

Dose / conc.:

15 600 mg/kg diet

Remarks:

L-lysine at 100% excess over controls. Basis: nominal in diet

Dose / conc.:

7 800 mg/kg diet

Remarks:

L-lysine at 50% excess over controls. Basis: nominal in diet

No. of animals per sex per dose:

11 control females, 14 females per treated group

Control animals:

yes

Details on study design:

Pilot projects
Two pilot studies were performed to determined if diets with excess of lysine support growth and health in non-pregnan female rats. Four of the experimental groups received diets with different excess lysine concentrations (50, 100, 500, 1000%). The body weight, behaviour and general apperance were determined for 4 wekks, before the animals were sacrificed and analyzed for alteration in organs or fat deposits.

A second pilot project was designed to determine if the chosen supplementation level of lysine would support pregnant rats so that delivery of viable offspring would be possible. Virgin Sprague Dawley rats were time mated by the supplier.
With the appearance of a seminal plug, the following morning was designated as the beginning of day 0. The animals were delivered to the university vivarium on day 0, weighed and randomly assigned to groups. Each of these groups were fed excess lysine diets: 50, 100 or 500%. Animals were fed ad libitum and food intake and spillage were monitored five times weekly from day 0 through day 20 of gestation. Body weight was measured three times weekly. General appearance of the animals was carefully noted throughout the course of pregnancy. On day 20, the animals were sacrificed, the uteri and ovaries were removed and the numbers of corpora lutea and foetuses plus resorption sites were compared to verify the number of resorptions. Placentas were weighed on a top loading balance after removal of membranes and a general examination for external malformations was completed. The foetuses were measured and sexed.

Main study
Virgin Sprague-Dawley rats were time mated at night by the supplier. Because it was determined in earlier phases of the study that in some cases the experimental diet was associated with reduced food intake, pair-fed control groups were necessary for this final phase of the investigation. Control group (n=11) was fed the control diet ad libitum, while the test groups received excess lysine diets (50, 100 or 500%). In addition, 7 seven pair-fed control animals were fed the control diet, but at the level recorded for the experimental animals that they were paired with. Food intake was monitored daily from day 0 until day 20 of gestation.
On day 20 of pregnancy, the necropsy procedure referred to above was completed. Approximately three-fourths of the foetuses from each litter were placed in Bouin’s solution for subsequent examination of any structural variations. The remaining one-fourth of the foetuses from each litter was placed in 95% ethanol for later processing with alizarin stain which facilitated skeletal examination. After two weeks’ fixation in Bouin’s solution, the foetuses were razor sectioned. The head, neck and lower trunk were serially sectioned with a razor blade and after opening the thoracic cavity; sagittal sections were made of the heart. The foetuses stained for approximately 24 hr with alizarin red stain and subsequently placed in increasing concentrations of glycerine were examined generally for skeletal defects and variations in ossification. The number of ribs and phalanges were counted.

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: through the course of pregnancy

BODY WEIGHT: Yes
- Time schedule for examinations: three times weekly

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: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: not described

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: fetal weight, fetal length and placental weight

Fetal examinations:

- External examinations: Yes: [3/4 per litter ]
- Soft tissue examinations: Yes: [3/4 per litter]
- Skeletal examinations: Yes: [¼ per litter ]
- Head examinations: Yes: [3/4 per litter]

Statistics:

One-way analysis of variance (ANOVA) and Duncan’s multiple comparison procedure

Historical control data:

not described

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decrease in matemal weight gain could be observed.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The lysine treatment groups und their pair-fed controls did not differ significantly from each other or the ad libitum control group in the amount of food consumed.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The highest supplemented group, Lysine-500, had a significantly smaller food efficiency ratio than any other group.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The lysine groups showed no significant differences in the mean number of early resorptions.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no statistically significant differences within lysine groups In the mean number of viable fetuses.

Details on maternal toxic effects:

Maternal toxic effects: yes

Details on maternal toxic effects:
Significantly lower maternal weight gain at 500% excess L-lysine over controls

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

other: body weight / L-lysine at 100% excess over controls.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The total fetal weight in each litter did not differ significantly among the lysine groups, their pair-fed controls or the control group.
Lysine-500 had significantly smaller average fetal weights than all other lyslne groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No significant differences were found in the ratio of males to fernales within the lysine groups.

External malformations:

no effects observed

Description (incidence and severity):

No obvious congenital malformations or abnormolilies were observed in any of the fetuses.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No skeletal variations were found consistently within any group although some variation in ossification of phalanges existed, particularly in the smaller fetuses; Sectioning of fetuses preserved in Bouin's solution showed that the fetuses were structurally normal; the few exceptions included all fetuses from a litter in group L-100 with larger livers than those of any other group.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: yes

Details on embryotoxic / teratogenic effects:
Fetuses of significantly smaller weight and length at 500% excess L-lysine over controls.

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.

Abnormalities:

no effects observed

Developmental effects observed:

not specified

none

Conclusions:

In summary, variable levels of lysine supplementation did not cause recognizable fetal malformations. However, diets highly supplemented with lysine may affect pregnancy through a decrease in matemal weight gain and fetal size.

Executive summary:

Gross examinaton of both fresh and preserved fetal specimens revealed no evidence that any level of lysine supplementation was teratogenic.
Likewise there was no evidence of declining litter size or increasing fetal resorptions with rising supplementation levels.
Food consumption and maternal weight gain are both continuous variables that provide an early indication of a substance's toxicity. There were no significant differences in the amount of food consumed wlthin the lysine groups.
The highest supplemented lysine group had significantly decreased maternal weight gains. The highly supplemented diets yielded reduced gain in maternal weight for each gram of food consumed (reduced food efficiency ratio). The basis for this relationship is uncertain but it may be due to the increased caloric wastage associated with metabolism of protein (amino acids) for energy as compared with either carbohydrate or fat utilization. With reduction in food intake and/or food efficiency in the highly supplemented animals, it is not surprising that total fetal weight and individual fetal size was affected. The lysine groups showed this trend in average, but not total fetal weight. Because L-500 had a significantly decreased average fetal weight yet did not consume significantly less diet, it appears that the decrease in fetal weight was related to reduced food efficiency. 
Data suggest, therefore, that maternal consumption of excess amounts of lysine may result in decreased fetal growth. The L-500 diet (500% excess lysine) relates to human supplementation with lysine of approximntely 35 g per day.
These levels of amino acid supplementation are clearly higher than those reported in human trials. For exarnple, the well-balanced and protein-adequatic North American diet contains about 5-8 g of lysine daily.

In conclusion, the results of this study suggest that variable levels of lysine supplementation do not cause grossly recognizable fetal malformations in rats. Lower supplementation levels of the amino acid do not appear to have any negative impact on maternal weight gain, fetal weight or other fetal parameters that may signify toxicity. Diets supplemented with a high amount of lysine (500% excess) appear to be associated with a lower than expected maternal weight gain and a significantly reduced fetal weight.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Justification for classification or non-classification

Based on the information available L-lysine HCl does not need to be classified (CLP regulation) for reproductive toxicity.