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Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Dose descriptor:
NOAEL
8 000 mg/kg bw/day
Additional information

Within the Vinasses category, data on toxicity to reproduction are only available for one subgroup of Vinasses, e. g. Vinasses, residue of fermentation. However, all Vinasses subgroups share a common origin and are therefore constituted of the same components determining their toxicological properties. Thus, read-across is performed based on a category approach (A detailed justification is attached in IUCLID section 13).

In an extended subchronic oral toxicity study with Vinasses, residue of fermentation (Appel, 2003), fertility parameters including sperm analysis and oestrus cyclicity were examined (Waalkens-Berendsen et al., 2003). The test material was fed ad libitum to groups of 5 male and female rats at 2, 6 and 15% in the diet for 13 weeks. The corresponding mean dose levels over the study period were 1000, 3000 and 8000 mg/kg bw/day in males and 1200, 3300 and 9600 mg/kg bw/day in females. A negative control group fed a plain diet, and a positive control group receiving additional ammonium (derived from (NH4)2S04); approx. 0.6% in diet, corresponding to 320 and 380 mg/kg bw/day in males and females, respectively) were included.

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 and the high-dose group were evaluated.

Parameters examined in males included testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology. Of all males of each group, the left cauda epididymis was weighed and epididymal sperm was derived, in which 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.

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.

No effects were observed on daily sperm production, sperm morphology, sperm motility parameters and the number of spermatozoa per gram testicular parenchyma. No statistically significant effects on epididymal sperm count were observed.

Based on the study results, the NOAEL for parental fertility parameter is considered to be 8000 mg/kg bw/day in males and 9600 mg/kg bw/day in females, which correspond to the highest dose levels tested.

There are no further studies available in which potential effects of Vinasses to reproduction have been investigated.

Various subgroups of Vinasses, namely (sugar) beet and (sugar) cane Vinasses, are listed in the Catalogue of feed materials (Annex to Commission Regulation (EU) No 242/2010). Hitherto, there is no information available indicating reproductive effects in livestock species.


Short description of key information:
Subchronic NOAEL (parental fertility, rat) = 8000 mg/kg bw/day (males)
Subchronic NOAEL (parental fertility, rat) = 9600 mg/kg bw/day (females)

Effects on developmental toxicity

Description of key information
Developmental and maternal NOAEL (rat): 3850 mg/kg bw/day
Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
3 850 mg/kg bw/day
Additional information

Within the Vinasses category, data on developmental toxicity are only available for one subgroup of Vinasses, e. g. Vinasses, residue of fermentation. However, all Vinasses subgroups share a common origin and are therefore constituted of the same components determining their toxicological properties. Thus, read-across is performed based on a category approach (A detailed justification is attached in IUCLID section 13).

Vinasses, residue of fermentation was tested for oral prenatal developmental toxicity in Wistar rats following OECD guideline 414 and in compliance with GLP (Wolterbeek, 2003). The test material was fed ad libitum to groups of 24 mated female rats at 2, 6 and 15% in the diet for 21 days (from fertilization (GD 0) until Caesarean section shortly before term (GD 21)). The corresponding mean dose levels over the study period were 1300, 3850 and 8700 mg/kg bw/day. A negative control group fed a plain diet, and a positive control group receiving additional ammonium (derived from (NH4)2S04); approx. 0.6% in diet, corresponding to a mean dose level of 370 mg/kg bw/day) were included.

As a consequence of the significantly reduced food consumption caused by the high nitrogen content of the test substance, body weights of dams were statistically significantly decreased from GD 7-21 in the ammonium control group and in the 15% test group as compared to the negative control group. Body weight changes were statistically significantly decreased in the ammonium control group and in the 15% test group between GD 0-3, 3-7 and 10-14 as compared to the negative control group. Furthermore, body weight change was statistically significantly decreased in the 6% test group between GD 0-3.

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 foetuses, 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.

Macroscopic findings did not differ among the groups.

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% test group. Foetal weight of the male foetuses was statistically significantly decreased in 15% test group. In the ammonium control group, male foetal weight was also decreased but the difference did not reach a level of statistical significance. No significant differences in foetal weight were observed between the ammonium control group and the 15% test group. These effects might be secondary to the observed decrease in maternal body weight.

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% test group statistical significant treatment-related effects were observed on variations in ossification of several skeletal bones as compared to the negative control group. Most probably, these effects were caused by ammonium since the effects observed in the 15% test group were similar to the effects observed in the ammonium control group.

In conclusion, under the conditions of the study feeding of Vinasses, residue of fermentation at dietary levels up to 6% (equivalent to a mean dose level of 3850 mg/kg bw/day) was tolerated without obvious signs of maternal and developmental toxicity. The effects 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 as were observed in the pregnant females and foetuses of the 15% test group (corresponding to 8700 mg/kg bw/day) were attributable to the ammonium present in the test material since these effects were also observed in the ammonium control group.

In this study, the NOAEL for maternal toxicity is considered to be 3850 mg/kg bw/day, while the LOAEL is allocated at 8700 mg/kg bw/day based on decreased body weight and food consumption. Similarly, due to the lack of effects in the offspring, 3850 mg/kg bw/day is considered the NOAEL for developmental toxicity. Based on decreased foetal weight, kinked ureters and various variations in ossifications, the dose level of 8700 mg/kg bw/day is identified as the LOAEL for developmental toxicity. It should be noted, however, that these effects are attributable to the ammonium present in the test material, in addition to maternal toxicity.

In an earlier study, the teratogenic and embryotoxic potential of two types of Vinasses were investigated in pregnant rabbits (van Eeken et al., 1983). Groups of 6 pregnant animals were administered the test materials by oral gavage at 0.25, 0.5 and 1.0 mL/kg bw/day from gestation day 1 to 15. Based on the respective density values, the corresponding dose levels were 320, 640 and 1280 mg/kg bw/day for the one Vinasses type, and 370, 740 and 1480 mg/kg bw/day for the other type. A control group of 20 pregnant rabbits treated with physiological saline was included. Clinical signs and body weight were recorded in maternal animals. On gestation day 16, the maternal animals were sacrificed and the uterine contents were examined macroscopically. Examinations included the number of implantations, resorptions, corpora lutea and live and dead foetuses.

No unscheduled mortalities occurred. One animal in the control group had diarrhoea on day 3 post-coitum. No further clinical signs were observed.

The results of the experiment showed that in all dose groups studied with both types of Vinasses, none of the parameters showed abnormalities compared to the control group. The authors concluded that none of the test materials tested could induce teratogenic or embryotoxic effects.

Based on the results of this study, the highest dose levels tested, e. g. 1280 and 1480 mg/kg bw/day, are considered the NOAEL values for both maternal toxicity and embryotoxicity in rabbits.

Justification for classification or non-classification

The available data on the potential toxicity to reproduction of the substance are conclusive but not sufficient for classification according to the DSD (67/548/EEC) and CLP (1272/2008/EC) criteria.