Multigenerational reproductive assessment of 4-methylimidazole administered in the diet to Hsd:Sprague Dawley SD rats

Abstract

The general population, including children and adolescents, is exposed to 4-methylimidazole (4-MI) in the diet. 4-MI is a by-product of caramel color manufacturing. It has been previously classified as a possible human carcinogen and displays potential reproductive toxicity. A follow up assessment of reproductive toxicity was conducted in rats utilizing the reproductive assessment by continuous breeding paradigm, in which multiple generations were exposed to 4-MI in diet at 750, 2500, and 5000 ppm. 4-MI exposure was associated with delays in preputial separation and vaginal opening, impairment in reproductive performance, and concomitant histopathological findings in the prostate, testis, and epididymis at 2500 and 5000 ppm. The Lowest Observed Adverse Effect Level for reproductive (based on prostate atrophy) and developmental toxicity (based on delays in preputial separation and vaginal opening) was 750 ppm, equivalent to approximately 50-60 mg/kg bw/day.

Keywords: 4-Methylimidazole; Development; Reproduction; Testicular toxicity.

Figure 1:

Schematic of the Reproductive Assessment by Continuous Breeding (RACB) Design Rats were administered 4-MI in the diet at 0, 750, 2500, or 5000 ppm ad libitum. The F0 adults were exposed during a 2-week prebreed exposure period, during cohabitation, gestation and lactation for the F1a, F1b, and F1c generations until necropsy. The F1c generation was exposed throughout life either indirectly via the mother during gestation and lactation and then by direct exposure to the dosed feed beginning on PND 28 until necropsy. The F2c generation was exposed to 4-MI via the mother during gestation and lactation. Reproductive and developmental performance was measured as described in the text. Additionally, a crossover mating was performed on the F0s following generation of the F1c to determine affected sex; during crossover mating, the 4-MI treated animals were crossed with naïve animals of the opposite sex.

Figure 2.

Mean body weights versus day for male and female rats at different developmental stages for control and dosed animals. An average of the litter mean weights are shown in (e)-(h). Due to apparent moribundity/mortality, F0 females in the 5000 ppm dose group were removed prior to mating for the C litter and are not shown in (c)-(h).

Figure 3.

Ventral prostate gland histopathology for F0 control and 5000 ppm 4-MI exposed males. Compared to the prostate from the control male, the prostate from the 4-MI treated male is smaller and the luminal contents are paler staining (compare control in A to F0 5000 ppm in B, 1x magnification). The luminal secretions in 4-MI treated prostates were often decreased, pale and occasionally flocculent (F0 5000 ppm in D, 10x; F, 40x) compared to the more densely eosinophilic secretory material in control prostates (C, 10x; E, 40x). Also, the epithelium lining the prostates of 4-MI treated males was often flattened and attenuated (F, 40x) when compared to normal control prostates lined by cuboidal to columnar epithelium (E, 40x). H&E.

Figure 4.

Histopathology of the germinal epithelium in the testis of F0 control and 5000 ppm 4-MI males (A, 20x, PAS/H). In an early-stage tubule (E), there is a focal area of germ cell dropout (marked with an oval), as well as apoptosis of germ cells (arrows). The mid-stage tubule (M) nearest the center of the image has depletion of elongating spermatids. A late-stage tubule (L) has depletion of elongating spermatids and spermatocytes, with decreased tubular diameter and disorganized germinal epithelium. Vacuoles are also present (*). The cauda of the corresponding epididymis (B) for the testis in panel A contains exfoliated germ cells and debris from the testis (40x, H&E). A stage XI tubule from an F1 generation male dosed with 2500 ppm 4-MI shows a cluster of retained mature spermatids at the luminal surface of the germinal epithelium (C, 40x). The mature spermatids (step 19 spermatids) should be released during stage VIII, and therefore should not be present in a stage XI tubule (step 11 spermatids), as seen in a control F0 male (D, 40x).