vinasses, residue of fermentation, depotassified

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: extended 90-day feeding study examining fertility parameters

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

In this study, the substance tested, Vinasses, residue of fermentation, has a chemical composition analogue to Vinasses, residue of fermentation, depotassified and therefore is used in an analogue approach. The analogue approach justification is described in the endpoint study summary. GLP-Guideline study - extended for fertility parameters.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

other: Annex to subchronic oral feeding study

Title:

Unnamed

Year:

2003

Report date:

2003

Reference 2

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. QA statement)

Remarks:

Inspectorate for Health Protection, Commodities and Veterinary Public Health; Ministry of Health, Welfare and Sport; The Netherlands.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Vinasses, residue of fermentation

IUPAC Name:

Vinasses, residue of fermentation

Constituent 2

Reference substance name:

932-215-9

EC Number:

932-215-9

IUPAC Name:

932-215-9

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 or test system 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 / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 2, 6 and 15%
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 1000, 3000 and 8000 mg/kg bw/d
Basis:
other: mean test substance intakes (approx.) in males

Doses / concentrations 3

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 generation)

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)

1

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.

2

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 * 10^6	Testicular parenchyma weight (g)	No. of spermatozoa per g testicular parenchyma * 10^6	Daily sperm production * 10^6
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