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EC number: 200-854-6 | CAS number: 75-25-2
Effect of the test substance on reproductive function in Swiss D-1 Mice.
The effect of the test substance on reproduction and fertility in Swiss CD-1 mice was assessed using a United States National Toxicology Program Reproductive Assessment by Continuous Breeding Protocol (RACB).
Male and female Swiss CD-1 mice were used for the study.
The RACB protocol is divided into four tasks. Task 1 is 14-day toxicity range-finder, Task 2 is a continuous breading phase, Task 3 is a 1-week mating trial and task 4 is an assessment of offspring. As the test substance had no effect on fertility Task 3 was not conducted as part of this study.
Following dose range-finder study (Task 1) the dose levels selected for Task 2 were 50, 100 and 200 mg/kg bw/day. For task 2 there were 20 male and female breeding pairs per treatment group and 40 male and female breeding pairs for control animals). Animals were dosed daily by gavage using corn oil as the vehicle for a 7-day pre-cohabitation and a 98-day cohabitation period. The last litter born during the holding period following the continuous breeding phase is reared by the dam until weaning, after which treatment is initiated by the same route and at the same concentration as during Task 2. These animals are used for assessment of second generation fertility (Task 4).
Endpoints assessed were clinical signs, parental body weight and average consumption of drinking water during representative weeks, fertility (number producing a litter/number of breeding pairs), litters per pair, live pups per litter, proportion of pups born alive, sex of live pups and the pup body weights immediately after birth. For Task 4 the last and generally the fifth litter was reared and weaned from the control and 200 mg/kg and kept to sexual maturity (74 ± 10 days) while housed by sex two per cage (maximum) and receiving the same treatment as their parents. At sexual maturity a male and female from different litters within the same treatment group are cohabited for 7 days and then housed singly until delivery. The endpoints assessed for this mating trial are the same as Task 2 with the addition of checking for the presence of a copulatory plug.
Six mice died or were sacrificed during this task. For two mice the cause of death could not be determined. The deaths of the other four mice were not considered related to treatment.
Fertility index was 100% for control and test substance groups (breeding pair was designated fertile if they produced at least one live or dead pup. The control and bromoform groups did not significantly differ in number of litters per pair, litter size, proportion of live pups and proportion of male pups, female pup weight or combined pup weight at birth. Male absolute pup weights in the 200 mg/kg test substance group were significantly less than control values but this difference was not significant after pup weights were adjusted for litter size. The cumulative days to litter were essentially identical across groups.
Body weights at delivery of dams receiving 200 mg/kg of test substance were consistently less than control values; these reduced body weights were statistically significant after delivering the first, second, fourth and fifth litters. During the Task 2 holding period, body weights of lactating dams did not differ among control and treatment groups.
Male and female adults were weighed on representative weeks during Task 2 and simultaneously, the water consumption per cage, or per pair, was monitored. Test substance treatment had no significant effect on body weight of males or females. The average daily consumption of drinking water was significantly increased in males and females treated with 50 mg/kg of test substance during weeks 2 and 10. In the 100 mg/kg dose group, male and female consumption was significantly increased during week 10. Consumption was also significantly increased in the 200 mg/kg dose group for males and females during weeks 2, 10, and 14.
Eleven F1 mice died during this task. The cause of death for four mice was not determined. The remaining deaths were not considered treatment related.
The postnatal survival among F1 pups in the 200 mg/kg bw/day test substance group was significantly lower compared to the control group. This decline was primarily attributed to three dams, which lost all their pups by postnatal day 4. One dam in the control group also lost her litter by postnatal day 4. Although these incidences are too low to evaluate statistically, they are consistent with a treatment effect on early maternal behaviour, early lactational failure, or postnatal developmental processes.
Test substance treatment had no apparent effect on fertility and reproduction in F1 mice. Some symptoms of general toxicity were noted in treated F1 mice. This included decreased male terminal body weights, increased adjusted liver weights in both sexes with accompanying hepatocellular cell degeneration and decreased adjusted kidney weights in both sexes. Epididymal ductal epithelium degeneration was noted in both control and bromoform-treated F1 males, and was not considered treatment-related.
Treatment with the test substance at up to 200 mg/kg bw/day had no effect reproduction or fertility in either the parental or F1generation. Treatment with test substance at 200 mg/kg treatment caused minimal to moderate histopathologic changes in the liver of second generation CD-1 mice.
The No Observed Effect Level (NOEL) for the study is considered to be 100 mg/kg bw/day.
Teratogenic effect of the test susbtance in Sprague-Dawley Rats.
The teratogenic potential of the test substance toSprague-Dawley ratswas assessed using a study design that pre-dated the OCED 414 guideline, but which nevertheless covered many of the endpoints included in the guideline.
Test substance was administered in corn oil by gavage to pregnant Sprague-Dawley rats from day 6 to day 15 of gestation at doses of 50, 100 or 200 mg/kg/day. A control group was administered corn oil only. Each group consisted of 15 rats and each rat was housed individually with free access to food and water.
On day 22 of gestation rates sacrificed and their viscera including the uteri were examined for pathological changes. The foetuses were removed, weighed individually and examined for viability and external malformations. Two pups from each dam were fixed in formalin for histological evaluation. Approximately two-thirds of the remaining live foetuses from each litter were placed in absolute ethanol for future staining of the skeleton with Alizarin red and subsequent examination for skeleton abnormalities. The rest of the foetuses were fixed in Bouin's fluid and studied for visceral changes.
Maternal rats were subject to gross pathological assessment and the liver, hear, brain spleen and one kidney were removed and weighed. The following tissue samples were taken from each animal and fixed: brain, heart, pituitary, thyroid, parathyroid, thymus, lungs, trachea and bronchi, bronchial node, liver, kidney, adrenal gland, spleen, skeleton muscle, peripheral nerve, salivary gland, skin, bone marrow, ovaries, uterus and bladder.
Maternal blood samples were taken for haematological and clinical chemistry assessments.
Following gross pathological examination a liver sample was taken from maternal animals for liver protein aniline hydroxylase (AH) and aminopyrine demethylase (APDM) activity.
Body and Organ Weights: There were no effects on maternal weight, liver, kidney or spleen weights resulting from treatment with test substance.
Foetal effects: There were no effects or litter size, incidence of resorptions, foetal weight or incidence of visceral anomalies resulting from treatment with test substance.
Histopathology: There were no dose related histological, changes in mother or foetuses resulting from treatment with test substance.
Haematology: Maternal haematological parameters assessed showed no affects from treatment with test substance.
Clinical Chemistry: Maternal clinical chemistry parameters assessed showed no affects from treatment with test substance.
Liver Enzymes: Maternal liver enzyme parameters assessed showed no affects from treatment with test substance.
Foetal toxicity: There was some of evidence of dose related increase in sternebra aberrations and observations of interparietal anomalies in foetuses at 100 and 200 mg/kg bw/day. These were considered to be the result of a foetal toxicity effect and not teratogenicity related.
The test substance was not considered teratogenic to Sprague-Dawley rats. The study No Observed Effect Level (NOEL) for teratogenicity was 200 mg/kg bw/day (highest dose administered).
There was some evidence of a foetal toxicity caused by the test substance. The study No Observed Adverse Effect Level (NOAEL) for foetal toxicity was considered to be 100 mg/kg bw/day.
There was some of evidence of dose related increase in sternebra aberrations and observations of interparietal anomalies in foetuses at 100 and 200 mg/kg bw/day. These were considered to be the result of a foetal toxicity and not teratogenicity related.
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