Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: extended 90-day feeding study examining fertility parameters
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study - extended for fertility parameters.
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
other: Annex to subchronic oral feeding study
Title:
Unnamed
Year:
2003
Report Date:
2003
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Principles of method if other than guideline:
Within a subchronic oral toxicity study, sperm analysis and oestrus cyclicity screening was performed.
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), (Liquid by product from the production of lysine and threonine by E. Coli K12)
- Physical state: dark brown viscous liquid
- Analytical purity: 100%
- Lot/batch No.: Not given (only one batch was produced; CP trial 13/14 Nov 2001)
- Expiration date of the lot/batch: 12 Apr 2004
- Storage condition of test material: 2-10 °C, in a dry place
- Composition: dry matter: 61.14%, water: 38.86%, total nitrogen: 6.22%, crude proteins: 38.87%, ammoniacal nitrogen: 4.18%, pH (solution 10%) 4.4, ashes: 2.78%, K: 0.17%, Na: 0.87%, Ca: n.d., Mg: n.d., Cl: 0.375%, SO4: 14.23%, P: 0.12%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Weight at study initiation: 174.8-215.8 g (males), 126.7-156.9 g (females)
- Age at study initiation: approx. 7 weeks
- Fasting period before study: no
- Diet (ad libitum): AIN-93G control diet from 4 days before study initiation on
- Water: ad libitum
- Acclimation period: the animals were delivered approximately 2 weeks before the start of the study

IN-LIFE DATES: 03 July 2002 - 15 Oct 2002

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: plain diet
Details on mating procedure:
No matings performed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Integrity of the test diets
In the first batch of test diets the following analyses were performed:
- dry matter (TNO SOP DASlLNCl069);
- crude protein (TNO SOPS LNCIlO1 en LNC102);
- crude fat (TNO SOP LNCl139);
- crude fibre (TNO SOP DASlLNCIO11);
- ash (TNO SOP DAS/LNCl131);
- Ca, P, Na, K (TNO SOP DAMlLSPl057);
- Cl (TNO SOP LNCl216).
Duration of treatment / exposure:
13 weeks of dosing
Frequency of treatment:
daily ad libitum
Details on study schedule:
No matings performed.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 2, 6 and 15%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 1000, 3000 and 8000 mg/kg bw/d
Basis:
other: mean test substance intakes (approx.) in males
Remarks:
Doses / Concentrations:
0, 1200, 3300, 9600 mg / kg bw / d
Basis:
other: mean test substance intakes (approx.) in females
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
other: additional ammonium ((derived from (NH4)2S04); approx. 0.6%) control group
Positive control:
0.6% ammonium (derived from (NH4)2S04)

Examinations

Parental animals: Observations and examinations:
see 90-day oral feeding study under IUCLID section 7.5.1: key, Appel, 2003, 13 weeks, Sirional E.Coli, RL1, Repeated dose toxicity: oral
Oestrous cyclicity (parental animals):
Vaginal smears to evaluate the oestrus cycle length and normality were made daily for about 3 weeks prior to sacrifice. Smears were made of all females, but only the smears of the control groups (A+B) and the high-dose group (E) were evaluated.
Sperm parameters (parental animals):
Of all males of each group left cauda epididymis was weighed and epididymal sperm was derived from the left cauda epididymis and the motility was measured. The cauda epididymal sperm reserves were enumerated. A smear of the sperm solution was stained and 200 spermatozoa of each male of the control groups and high-dose group were analysed.
At necropsy the left testis was frozen for later evaluation. Testis was weighed and afterwards homogenized. The homogenization resistant sperm heads were enumerated in the control groups and high-dose group.

Parameters examined in males:
testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology.
Litter observations:
Not performed
Postmortem examinations (parental animals):
see IUCLID section 7.5.1
Postmortem examinations (offspring):
Not performed
Statistics:
Oestrus cyclicity was evaluated by Fisher's exact test (number of acyclic animals and number of animals with prolonged oestrus period) or ANOVA followed by Dunnett's multiple comparison tests (number of cycles per animal, mean cycle length). Sperm parameters were evaluated by ANOVA followed by Dunnett's 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).

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The number of acyclic females, the mean cycle length, the number of cycles per animal and the number of females with prolonged oestrus period were comparable in all groups.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No effects on sperm motility parameters were observed.
No statistically significant effects on epididymal sperm count were observed.
No effect on the number of spermatozoa per gram testicular parenchyma was observed.
No effect on daily sperm production was observed.
No effect on sperm morphology was observed.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental fertility parameters
Effect level:
8 000 mg/kg bw/day
Based on:
test mat.
Remarks:
equivalent to 15% in the diet
Sex:
male/female
Basis for effect level:
other: No effects on epididymal sperm motility, epididymal sperm count, testicular sperm count, daily sperm production and sperm morphology.
Dose descriptor:
NOAEL
Remarks:
parental fertility parameters
Effect level:
9 600 mg/kg bw/day
Based on:
test mat.
Remarks:
equivalent to 15% in the diet
Sex:
female
Basis for effect level:
other: No effects on oestrus cyclicity.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Oestrus cyclicity

 

Control Group A

Control Group B

High dose group

Number of females evaluated

10

10

10

Number of acyclic females

1

1

0

Mean cycle length (days)

5.44

5.22

4.90

Number of animals with

prolonged oestrus period

0

0

1

Number of cycles per animal

9

9

10

 

Table 2: Summarizing data for sperm motility analysis (mean values; only data from control groups and highest dose shown)

Group

Motile cells (%)

Static cells (%)

Progressive cells (%)

VAP

(μm/s)

VSL

(μm/s)

VCL

(μm/s)

STR

(%)

LIN

(%)

ALH (μm)

BCF

(Hz)

Control A

50.9

49.1

32.30

101.98

66.24

244.42

58.20

24.5

16.24

23.80

Control B

61.8

38.2

47.0

123.26

81.91

299.25

66.40

27.5

18.64

23.37

High-dose

43.33

56.67

28.78

108.39

72.23

244.47

52.26

26.11

16.89

21.60

 

Table 3: Summarizing data for epididymal and testicular sperm count (mean values)

Group

Cauda epididymal weight (g)

Number of spermatozoa per mL * 106

Testicular parenchyma weight (g)

No. of spermatozoa

per g testicular parenchyma * 106

Daily sperm production * 106

Control A

0.26

17.99

1.35

299.04

49.02

Control B

0.26

17.96

1.43

315.84

51.78

High-dose

0.25

12.88

1.28

283.53

46.48

 

 

Applicant's summary and conclusion