Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October – December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
other: final report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
no guideline required

Results and discussion

Any other information on results incl. tables

Toxicokinetics evaluation

Acute studies

The test substance, Direct Brown 115/116, was applied to laboratory animals (rat, rabbit, guinea pig) during studies with different way of entry into organism (e.g. stomach, skin and eye).

After single oral administration of the test substance to rats, clinical signs of intoxication as diarrhoea and languor were recorded (results from the 1990 provided by Sponsor). The value of LD50 was established as 8.683 (7.947 – 9.489) g/kg. Internet search reported the value of LD50 greater than 2232 mg/kg. After single dermal administration of the test substance to rats, no clinical signs of intoxication were observed (results from the 1990 provided by Sponsor). The LD50 was established higher than 5 g/kg. No systemic toxicity was observed, so the substance probably did not enter the organism through the skin. According this result can be predicted that the test substance is non-toxic after single dermal application.

According to result of acute eye irritation/corrosion test and skin irritation test (results from internet search) the test substance is not irritant to the eye and skin of rabbit. The substance elicited negative sensitising response in guinea pig maximisation test.

 

Repeated toxicity

Significantly decreased mean of body weight was detected in animals of both sexes at the highest dose level. In some time weight period the body weight gain was decreased by more than 10% compared to control so this effect could be regarded as toxicologically significant. In males at the highest dose level also decrease of food consumption was recorded.

Biochemical examination showed increased value of protein total and albumin in both sexes (irreversible in females).

Histopathological examination showed findings in rectum (infiltration of mucosa and/or submucosa, brown pigment in mucosa and focal erosion) and intestines (brown pigment in mucosa, epithelium coloured by the test substance). Occurrence of microscopic findings in rectum and/or intestine probably related with place of application (digestive tract). Presence of brown pigment in mucosa in rectum did not disappear even after the end of recovery period.

During the histopathological examination also serious finding on skeletal muscle in males and females (atrophy of muscle fibres) was found out.

During the functional observation markedly decreased number of upstanding in males at the middle and highest dose levels and in females at the highest dose level was recorded. Biochemical examination showed decreased value of creatinine in males in all treated groups and in females at the highest dose level. Also increased activity of AST and ALT in males and females was detected. These values (values of creatinine, values of activity of AST and ALT) were out of historical control limits. Decrease of these parameters related to serious damage of muscular matter which was confirmed during the functional observation and by the histopathological examination.

The value of NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY was established as 250 mg/kg body weight/day both for MALES and FEMALES. The value of NOAEL was determined on the basis results of histopathological examinations and biochemical examinations. 

 

Reproduction and development

During the examination of microscopical structure of reproductive organs toxicologically significant findings in males at the highest dose level were found out (in testes: tubular dystrophy, tubular atrophy, brown pigment in interstitium; in epididymis: germ cells and/or cell debris in lumen of tubules, mononuclear infiltration of epithelium and/or interstitium, brown pigment in interstitium, oligospermia or azoospermia). Decreased absolute weight of prostate gland+seminal vesicles and epididymis was recorded in males at the highest dose levels. Also the higher number of males with decreased sperm motility was recorded there.

In females only histopathological findings in uterus which related to females gravidity or oestrus cycle were found out. 

Number of females achieving pregnancy was seriously affected by the test substance treatment - in females at the highest dose level was the lowest. Number of implantations was decreased at the middle and highest dose level. One female at the highest dose level with abort was recorded.

Male and female fertility index and gestation index at the highest dose level were decreased markedly. Post-implantation loses and post-natal loses were increased at the highest dose level.

The concentration of thyroid hormone thyroxine (T4) in males and pups was not significantly changed.

The mean of number of pups and sex ration (M/F) was lower at the highest dose level compared to control group. In pups at the highest dose level the mean body weight and mean weight of litter were markedly decreased. In one female at the highest dose level the number of pups was not determined by the reason of cannibalism of pups immediately after parturition. Also in one female all pups died during the lactation period. During the macroscopic examination of these pups empty stomach (without milk) in five pups and haematoma in head in two pups were recorded.

No presence of nipples in male pups was recorded and anogenital distance in treated male and female pups was not significantly affected.

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION and DEVELOPMENT was established as 500 mg/kg body weight/day. The value of NOAEL was determined on the basis results of histopathological examinations, examination reproduction parameters. 

Applicant's summary and conclusion

Executive summary:

According to current valid guidance the test substance with the result of LD50 in acute oral test higher than 2000 mg/kg is considered as non-toxic after single oral application. Direct Brown 115/116 did not penetrate into the organism through the skin after single application.

The test substance is considered to be non-irritating to the eye and skin of rabbit.

The test substance elicited negative sensitising response in guinea pigs.

According to results of acute exposition of the test substance Direct Brown 115/116 can be expected, that the test substance did not penetrate through the skin and digestive tract.

The systemic toxicity after repeated oral administration of the test substance was observed. In some time weight period the body weight gain of high dosed animals was decreased by more than 10% compared to control so this effect could be regarded as toxicologically significant. During the histopathological examination serious finding on skeletal muscles in males and females (atrophy of muscle fibres) was found out. A related changes in biochemical parameters as changed values of creatinine, values of activity of AST and ALT (out of historical control limits) were recorded. The atrophy of muscle fibres was confirmed during the functional observation (markedly decreased number of upstanding).

Test substance negatively influenced reproduction parameters of males and females. Direct Brown 115/116 affected sperm motility, weight of reproductive organs and microscopical structure of reproductive organs of males. Toxicologically significant findings were recorded in males at the highest dose level during the examination of microscopical structure of reproductive organs. A related decrease of number of females achieving pregnancy was observed after test substance treatment.

Development of pups was not influenced by the test substance administration.

According to results, the test substance after repeated administration affects not only tissues at application site, but is absorbed from the digestive tract into the bloodstream and affects other organs – skeletal muscles. Next target where the substance acts are the reproductive organs of males (penetrates into the testes), but the test substance Direct Brown 115/116 did not penetrate through the placental barrier.

No data about metabolism and excretion of the test substance were found.