Safety evaluation of certain food additives - ipcs inchem

CALCIUM LIGNOSULFONATE (40–65) 21
2000 mg/kg bw per day (groups 1, 2, 3 and 4, respectively). Each group was divided
into subgroups: allocation A of groups 1–4 consisted of 20 animals per sex per group
and was treated for 90 days (main animals). Groups 1 and 4 had additional recovery
animals that were kept for an additional 28 days without treatment (10 animals per
sex per group; allocation B). Allocation C of groups 1–4 consisted of six satellite
animals per sex per group and was used for investigation of changes in primary
immune response using the sheep red blood cell assay (functional testing of
immune response). In addition, the distribution of the leukocyte subpopulation was
studied after 13 and 17 weeks in allocation B animals using flow cytometry. The diet
was prepared once weekly and offered to the animals ad libitum. Dietary
concentrations were adjusted each week based on body weights and food
consumption. The diet was analysed for accuracy, homogeneity and stability;
analytical results showed that the diets were accurately prepared and that calcium
lignosulfonate (40–65) was stable in the diet and distributed homogeneously.
Animals were observed for moribundity and mortality twice daily and for clinical
signs once daily. During weeks 13 and 17, animals were subjected to a functional
observational battery using a modified Irwin screen. Hindlimb and forelimb grip
strength as well as locomotor activity were determined. Body weights and food
consumption were determined weekly. An ophthalmoscopic examination was
carried out before treatment, near scheduled termination and after the recovery
period. Detailed haematological and clinical laboratory examinations were
performed after 2, 6, 13 and 17 weeks using blood from the retro-orbital plexus. At
the same time points, urinalysis was performed and faecal pH was measured.
Sperm count, motility and morphology were assessed after 13 and 17 weeks. Sperm
samples used for motility measurements were obtained from the left caudal
epididymides of the animals. For assessment of sperm motility, 100 sperm were
counted. Morphology was determined after fixation; 500 sperm were
microscopically evaluated. Sperm and spermatid counts were determined in the left
caudal epididymides and left testis after homogenization by manual counting. The
estrous cycle was investigated during weeks 10–12 and 15–16. All animals were
subjected to a detailed gross necropsy after termination. Liver, kidneys, adrenals,
gonads, testes, epididymides, thymus, spleen, brain, thyroid/parathyroid, ovaries,
uterus and heart were weighed. These tissues plus others specified in the OECD
guidelines were preserved for histopathological examination. The control and highdose
groups were subjected to histopathological examination. Based on the chronic
inflammation of the rectum seen in the high-dose animals in the 4-week feeding
study (Weber & Ramesh, 2005), the rectum of all animals was subjected to
histopathological investigation. Additionally, the mesenteric lymph nodes and the
kidneys of the low- and mid-dose groups were evaluated by histopathology because
of changes observed in the respective organs of the high-dose animals.
Target dose levels were achieved. Males consumed 0.8%, 0.6% and
1.13% of the target dose levels of 500, 1000 and 2000 mg/kg bw per day,
respectively. Females consumed +1.3%, +1.8% and +2.0% of the target dose levels
of 500, 1000 and 2000 mg/kg bw per day, respectively. Two males were prematurely
sacrificed (low- and mid-dose groups) for ethical reasons. One animal had an
intestinal intussusception as revealed at necropsy, and the other had a markedly
enlarged eye; both events were not considered to be treatment related. Other