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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
Study period:
20 Oct - 22 Nov 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(adopted 22 January 2001)
Deviations:
no
Principles of method if other than guideline:
The objective of the study was to provide data on the possible effects of the test substance Sirional E.Coli (LYS + THR) on the pregnant female rat and on the development of the embryo and fetus. Female rats were given the test substance through the diet from fertilization (gestation day [GD] 0) until Caesarean section shortly before term (GD 21). Furthermore, animals in an additional control group were fed diets supplemented with ammonium to a level similar to that in the high-dose group (approximately 0.6%).
GLP compliance:
yes (incl. certificate)
Remarks:
Inspectorate for Health Protection, Commodities and Veterinary Public Health; Ministry of Health, Welfare and Sport; The Netherlands.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Sirional E. Coli (LYS + THR)
- Other name (as cited in study report): liquid by-product from the production of lysine and threonine by E. Coli
- Physical state: dark brown visquous liquid
- Analytical purity: not relevant (complex mixture used as feed material)
- Batch number: CP trial 13-14 Nov 2001
- Storage conditions: Refrigerator (2-10 °C)
- Expiry date: 01 Apr 2004

A certificate of analysis of test substance was provided. The analytical results are given in Table 1 below.

- Name of control substance (as cited in study report): Ammonium sulfate
- Supplier: Merck
- Batch number: A331217-203
- Expiry date: 31 Aug 2006

Test animals

Species:
rat
Strain:
other: Wistar outbred (Crl:(WI)WU BR)
Details on test animals and environmental conditions:
For the oral prenatal developmental toxicity test with PL73 E.Coli (LYS) and PT73 E.Coli (THR) in rats (TNO study 4840) and the present study a total of 340 female (about 10 weeks old) and 115 male (about 11 weeks old) Wistar outbred (Crl:(WI)WU BR) rats were obtained from a colony maintained under SPF conditions at Charles River Deutschland, Sulzfeld, Germany.
For this study, 130 females and 45 males were used. At the beginning of the mating period the females were at least 12 weeks old.

Upon arrival on 9 October 2002, the rats from this study were taken to a quarantine room and checked for overt signs of ill health and anomalies. During the quarantine period (in total 5 days) serological controls of their microbiological status were conducted (in 5 randomly chosen animals of study 4840) on 10 October 2002. After the results of serology indicated an acceptable microbiological status (on 14 October 2002), the animals of both studies were released for use in the study and further acclimatised until the start of the mating period on 20 October 2002.
Mated females were distributed over the 5 experimental groups in such a way that the animals from the same day of pregnancy were equally distributed over the groups. Females mated by the same male were placed in different groups.

The room was ventilated with about 10 air changes per hour and was maintained at a temperature of 19-25 °C. The relative humidity was between 30-70% except for some short periods on 18, 21, 22, 23, 25, 27 and 28 October 2002 and on 1, 4, 8, 13, 18 and 22 November 2002 when humidity exceeded 70%, reaching a maximum of 100%. Lighting was artificial with a sequence of 12 hours light and 12 hours dark.

Mated females were housed individually.
- Diet (ad libitum): rodent diet (Rat&Mouse No. 3, Breeding Diet, RM3) obtained from SDS Special Diets Services, Witham, England.
- Water (ad libitum): tap water

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: control diet AIN-93G
Details on exposure:
Sirional E.Coli (LYS + THR) was administered through the diet at constant concentrations (0,2, 6 and 15%) from fertilization (GD 0) until Caesarean section shortly before term (GD 21). Furthermore, animals in an additional control group were fed diets supplemented with ammonium to a level similar to that in the highdose group (approximately 0.6%).

PREPARATION OF DOSING SOLUTIONS:
The test substance was incorporated in the AIN-93G diet by mixing in a mechanical blender. Since the amount of food left from the sub-chronic (13-weeks) toxicity study with Sirional E.Coli (LYS + THR) (TNO Study 4619104, diets prepared on 30 September 2002 and stored < -18°C) was enough for the present study the experimental diets prepared for study 4619104 were also used in this study. Only the AIN-93G control diet used before the start of the mating period (see section 2.3.3) was prepared fresh for the present study on 17 October 2002 (because it concerns only a batch of control diet no analyses was performed).

Three days before the start of the mating period, the rats were fed the AIN-93G control diet, in order to acclimatise them to the purified diet. Until initiation of treatment, the rats were fed a modified AIN-93G purified rodent diet. The feed was provided as a powder, in stainless steel cans, covered by a perforated stainless steel plate that serves to prevent spillage. The feed in the feeders was refreshed approximately twice per week.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: until evidence of copulation (vaginal smears) was found
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

One female was caged with one male for mating. Every consecutive morning during the mating period, vaginal smears were made to ascertain copulation by detection of sperm cells in the smear. Upon evidence of copulation, positive females were housed individually and replaced by another nulliparous female until all females were placed with a male. The day a sperm-positive smear was detected was considered as GD 0.
Duration of treatment / exposure:
The test substance was fed from fertilization (GD 0) until Caesarean section shortly before term (GD 21).
Frequency of treatment:
ad libitum feeding
Duration of test:
21 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 2, 6 and 15%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 1300, 3850 and 8700 mg/kg bw/d
Basis:
actual ingested
measured mean test substance intakes
Remarks:
Doses / Concentrations:
370 mg/kg bw/d
Basis:
actual ingested
measured mean control substance (ammonium) intake in second control group
No. of animals per sex per dose:
The study comprised five groups of 24 mated female rats each, viz. one control group kept on control AIN-93G diet, one control group kept on AIN-93G diet supplemented with about 0.6% ammonium (derived from W4)2S04) and three test groups Sirional E.Coli (LYS + THR).
Control animals:
yes, plain diet
other: Ammonium control group (receiving plain diet mixed with 0.6% ammonium sulfate as control substance)
Details on study design:
- Dose selection rationale: The dose levels used in this study were based on the results of a (non-GLP) tentative range finding study (TNO Study 4619102) and were the same as used in a sub-chronic (1 3-weeks) toxicity study (TNO Study 46 19/04).

Examinations

Maternal examinations:
At Caesarean section females and fetuses of all groups were macroscopically examined. Fetuses, placentas, reproductive organs were weighed. Fetuses were further processed for fetopathological examination.

CLINICAL OBSERVATIONS: Yes
Each female 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 minimise 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
- Time schedule for examinations: Body weights of each mated female were recorded on GD 0, 3,7, 10, 14, 17 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The quantity of food consumed by each mated female was measured over GD 0-3, 3-7,7-10, 10-14, 14-17 and 17-21 by weighing the feeders.
The intake of the test substance per kg body weight per day was calculated from the nominal dietary concentration of the test substance, the food consumption and the body weight of the animals.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: Maternal tissue showing macroscopic abnormalities were removed and fixed
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

The uteri (including the fetuses), ovaries and placentas of all females killed on GD 21 were examined for the following parameters
- number of corpora lutea
- number of implantation sites
- number of early and late resorptions
- 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 foetuses
- Soft tissue examinations: Yes: half of the fetuses per litter
- Skeletal examinations: Yes: half of the fetuses per litter
- Head examinations: No data

The fetopathological examinations were initially restricted to all fetuses of the animals of the control groups (A and B) and the high-dose group (E). Since statistical significant effects were observed on visceral and skeletal observations in the 15% Sirional E.Coli (LYS + THR) group, after consultation with the sponsor, visceral and skeletal examinations were extended to the fetuses of the intermediate groups (2 and 6% Sirional E.Coli (LYS + THR) groups).
Since the number of fetuses of dam 625 (blind number 1 195) of the 15% Sirional E.Coli (LYS + THR) group and of dam 53 1 (blind number 1227) of the 2% Sirional E.Coli (LYS + THR) group) at fetopathological examination was not the same as observed at Caesarean section, it was concluded that
during processing of the fetuses a mistake was made. For this reason, the fetopathological observations of the fetuses of dam 625 (blind number 1195) were excluded from the results and the fetuses of dam 531 (blind number 1227) were not examined.
During the fetopathological examination the observer was unaware of the dose group of the fetuses.
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 late 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 performed to attempt to identify any specific dose-related effect that may have occurred. As a level of significance was considered: p < 0.05
Indices:
For each group the following parameters were calculated:
- Female fecundity index = (number of pregnant females / 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 / number of females pregnant) X 100
- Sex ratio = (number of live male fetuses / number of live fetuses) X 100
Historical control data:
no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: at 8700 mg/kg bw/day

Details on maternal toxic effects:
Body weights were statistically significantly decreased from GD 7-21 in the ammonium control group and in the 15% Sirional E. Coli (LYS + THR) group as compared to the 0% Sirional E. Coli (LYS + THR) group. Body weight changes were statistically significantly decreased in the ammonium control group and in the 15% Sirional E. Coli (LYS + THR) group between GD 0-3, 3-7 and 10-14 as compared to 0% Sirional E. Coli (LYS + THR) control group. Furthermore, body weight change was statistically significantly decreased in the 6% Sirional E. Coli (LY S + THR) group between GD 0-3. No differences in body weight and body weight changes were observed between the ammonium control group and the 15% Sirional E. Coli (LYS +THR) group.

Food consumption (expressed as g/kg body weight/day) was statistically significantly decreased in the ammonium control group between GD 0-3 and 7-21 and in the 15% Sirional E. Coli (LYS + THR) group between GD 0-17. Food consumption (expressed as g/animal/day) was statistically significantly decreased in the ammonium control group and in the 15% Sirional E. Coli (LYS + THR) group during the entire gestation period. The differences in food consumption between the ammonium control group and the 15% Sirional E. Coli (LYS + THR) group were less pronounced than between the 0 and 15% Sirional E. Coli (LY S +THR) groups.

Each group comprised of 24 mated females. Sixteen, 19, 20, 18 and 19 females of the control, ammonium control, 2.0,6.0 and 15% Sirional E. Coli (LYS + THR) group, respectively, appeared to be pregnant (at Caesarean section). In the ammonium control group and in the 2% and 6% Sirional E. Coli (LYS + THR) groups, respectively, one, two and one females, respectively, had no viable pups. Consequently, 16, 18, 18, 17 and 19 females of the control, ammonium control, 2.0, 6.0 and 15% Sirional E. Coli (LYS + THR) group, respectively, had viable fetuses.

No differences were observed in the female fecundity index and gestation index among the groups. No differences were observed in the number of corpora lutea, implantation sites, pre- and post implantation loss, live and dead fetuses, resorptions and sex ratio among the groups.
Parental necropsy did not reveal any difference for the mean weight of the gravid uterus, the empty uterus weight or the ovaries weight between the control groups and the treatment groups.

Carcass weight and net weight change from day 0 were statistically significantly decreased in the ammonium control group and in the 15% Sirional E. Coli (LYS + THR) group as compared to the 0% E. Coli (LYS +THR) control group. No differences in carcass weight and net weight change were observed between the ammonium control group and the 15% Sirional E. Coli (LYS + THR) group. Macroscopic findings did not differ among the groups.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
3 850 mg/kg bw/day
Based on:
test mat.
Remarks:
equivalent to 6% in the diet
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
8 700 mg/kg bw/day
Based on:
test mat.
Remarks:
equivalent to 15% in diet
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
3 850 mg/kg bw/day
Based on:
test mat.
Remarks:
equivalent to 6% in the diet
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Effect level:
8 700 mg/kg bw/day
Based on:
test mat.
Remarks:
equivalent to 15% in diet
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: at 8700 mg/kg bw/day

Details on embryotoxic / teratogenic effects:
No treatment related effects were observed for foetal external- and placental observations and weights. Foetal weight of all viable foetuses and of the female foetuses was statistically significantly decreased in the ammonium control group and in the 15% Sirional E. Coli (LYS + THR) group. Foetal weight of the male foetuses was statistically significantly decreased in 15% Sirional E. Coli (LYS+THR) group. In the ammonium control group, male foetal weight was also decreased but the difference did not reach a level of statistically significance. No significant differences in fetal weight were observed between the ammonium control group and the 15% Sirional E. Coli (LYS +THR) group.

No visceral malformations were observed. Observed visceral anomalies and variations were considered not to be adversely related to treatment.

Observed skeletal anomalies and variations of foetal skeletons were considered not to be related to treatment. In the ammonium control group and in the 15% Sirional E. Coli (LYS + THR) group statistical significant treatment-related effects were observed on variations in ossification of several skeletal bones as compared to the 0% Sirional E. Coli (LYS + THR) group. Most probably, these effects were caused by ammonium since the effects observed in the 15% Sirional E. Coli (LYS + THR) group were similar to the effects observed in the ammonium control group.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Compared to the 0% E. Coli (LYS + THR) group, in the 15% Sirional E. Coli (LYS + THR) group statistically significant effects were observed on body weight, body weight change, food consumption, carcass weight and net weight change from day 0, foetal weight, visceral variations (kinked ureters) and various variations in ossifications. Since no (or to a smaller extent) statistically significant differences were observed between the ammonium control group and the 15% Sirional (E. Coli (LYS + THR) group, in was concluded that the effects observed in the 15% E. Coli (LYS + THR) group were attributable to the ammonium present in E. Coli (+ THR).

Applicant's summary and conclusion