The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program (NTP). The findings may or may not have been peer reviewed and were not evaluated in accordance with the Explanation of Levels of Evidence for Reproductive Toxicity criteria (see http://ntp.niehs.nih.gov/ntp/htdocs/levels/09_3566_NTP_ReproTOX_R1.pdf or with the Explanation of Levels of Evidence for Developmental Toxicity criteria (see http://ntp.niehs.nih.gov/ntp/Test_info/NTP_DevTox20090507.pdf) established by the NTP in March 2009. The findings and conclusions for this study should not be construed to represent the views of the NTP or the US Government.
To examine the potential developmental and reproductive effects, 3,3',4,4'-Tetrachloroazobenzene (TCAB) was administered daily via oral gavage at doses of 0, 1.0, 3.0, and 10.0 mg/kg/day to groups (n=20 male and 20 female) of Sprague-Dawley rats for over two generations. The F0 generation (Task 2) began exposure as adults and they were bred continuously to produce F1a, F1b, and F1c pups. The F1c animals were raised and bred continuously to produce F2a, F2b, and F2c pups (Task 4, F1 generation). Significant reductions were observed in the number of F1 pups/litter at all dose levels except 1.0 mg/kg/day males. An Outbreeding Cohabitation was conducted between 10.0 mg/kg/day F0 males and naïve females and 10.0 mg/kg/day F0 females and naïve males.
Mean body weights exhibited a dose-related decrease in both the F0 and F1 males and females. Significant differences of 4-21% were noted in the F1 generation more often in the 10.0 mg/kg/day males and 3.0 mg/kg/day females. These decreases in body weights are at least partially related to a decrease in feed consumption when expressed as g/animal/day. When expressed as g/kg body weight/day, the decreases generally became smaller and increased in the case of F0 females.
The pregnancy index was not affected in the F0 cohabitation but by the F1 cohabitation there were significant decreases in the pregnancy index in all treated groups. The F1 pregnancy index was accompanied by an increase of 12-39% in the cumulative days to litter.
In the F0 cohabitation, the average number of litters per pair was comparable to controls but the average number of live pups (male, female and combined) per litter was decreased for all treated doses except for males at 1.0 mg/kg/day. These decreases were correlated with decreased mean body weights and adjusted weights for the male, female and combined weights at 10 mg/kg/day and adjusted weights at 3.0 mg/kg/day. Similar findings were observed in the F1 cohabitation with the additional findings of a decrease in the number of average litters per pair at 3.0 and 10.0 mg/kg/day.
The Task 3 Outbreeding indicated that TCAB was a female reproductive toxicant. There were no adverse effects when the naive females were cohabited with treated males but when the treated females were cohabited with the naive males there were decreased number of live pups per litter (significant for male pups only), and decreased live and adjusted weights (significant for combined live weight, and female and combined adjusted weight).
The development of the pups suggests that TCAB produced some neurotoxic problems. There were decreases in landing foot splay of 9-25% in the 3.0 and 10.0 mg/kg/day males and of 16-21% in the 3.0 and 10.0 mg/kg/day females; in forelimb grip strength of 24-43% in the 3.0 and 10.0 mg/kg/day males and females; and in hindlimb grip strength of 35% in the 10 mg/kg/day males.
No adverse findings were noted upon gross necropsy for the F0 and F1 adults, or F1 weanlings but there were numerous changes noted in absolute and relative organ weights, which were not consistent over the three groups of animals. Changes were noted in the liver, kidney, spleen, thymus, brain, testes and right epididymis. Sperm analysis of the F0 males revealed no changes. Sperm analysis of 10.0 mg/kg/day F1 males revealed decreases of total spermatid per testis by 26% and total sperm per cauda by 32%. An increase of 7-9% in the number of motile cells was observed in the 3.0 and 10.0 mg/kg/day F1 males. Microscopic findings noted at 10.0 mg/kg/day included minimal retention of Step 19 spermatids in both F0 and F1 males, chronic nephropathy in the F0 and F1 females, diffuse minimal hematopoietic cell proliferation in the spleen in the F0 males and females, minimal to mild lymphocytic depletion of the thymus in the F1 males and females, bile duct epithelial proliferation, and formation of granulomas (in one control and 9/14 Group 4 females) in the F1 females.
Based on conditions in this study, tetrachloroazobenzene resulted in reproductive toxicity at all dose levels as decreased pup body weights and number of pups; neurotoxicity at 3.0 and 10.0 mg/kg/day as evidenced by decreased male and female forelimb grip strength and landing foot splay, and general toxicity at all dose levels as evidenced by decreased parental body weight and feed consumption, and microscopic findings at 10.0 mg/kg/day in the liver, testes, spleen, kidney, and thymus. A no-observable-adverse effect level (NOAEL) was not reached in this study.