Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL = 15 mg/kg

LOAEL = 40 mg/kg bw

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
15 mg/kg bw/day
Study duration:
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Only a screening test on OECD 422 is available for Direct Violet 051. Information coming from this study are therefore not sufficient for classification purposes. For this reason data on Similar Substace 01 have been taken into account. The read across approach can be considered as reliable and adequate for the purpose; details and explanations are detailed in the report attached to the IUCLID section 13.

In the key study, three groups of ten male and ten femalerats received the substance at doses of 5, 15 or 40 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, 1water, at the same volume-dose as treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis (T4), estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.


There were no treatment related adverse effects of treatment on clinical condition, sensory reactivity, grip strength, body weight gain, food intake, haematology, blood chemistry or organ weight measurements in males and females. 

The assessment of motor activity scores indicated that males receiving 40 mg/kg/day were slightly less active than the control males.

Estrous cyclicity was unaffected by the administration of the substance at all dose levels. Out of 10 females that mated in Group 4 (40 mg/kg/day), only five achieved pregnancy

Hematological examination for males revealed, when compared with controls, slightly low hematocrit counts attaining statistical significance at 15 or 40 mg/kg/day. A slight increase in mean cell volume (attaining statistical significance) was observed in females treated at 40 mg/kg/day. In addition a decrease of the number of lymphocytes was seen in females at 40 mg/kg bw.

Biochemical evaluation revealed no treatment-related findings.

At the macroscopic examination of the adult animals no treatment related effects were observed. Pale areas in heart and kidneys could be related to minralization as observed during histopathological examnination.

At the microscopic examination of the adult animals changes related to treatment with the substance were seen in males at 40 mg/kg bw. These findings included minimal to slight degeneration/atrophy in the testes in 4/9 males treated at 40 mg/kg/day. A minimal increase in luminal cell debris in the epididymides was seen in the majority of males treated at 40 mg/kg/day; this was also present in one animal given 15 mg/kg/day,but the effect remained within historical control ranges (0 -20%).  In three animals given 40 mg/kg/day, the luminal cell debris were associated with minimal to moderate interstitial inflammatory cell infiltrate. 

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 male and female offspring.

The pregnancy rate in females at 40 mg/kg bw was 50% (leading to 4/10 viable litters). This reduction in fertility at 40 mg/kg/day did not show a correlation between the findings in the testes and those females which were found not pregnant. Therefore it remains unclear if the fertility was attributed to the males or the females treated with the substance. Apart from the reduction in fertility, there was no systemic toxicity detected in females treated with the substance.

The clinical condition of the offspring, litter size, offspring survival and sex ratio were unaffected by parental treatment.

Ano-genital distances of both male and female offspring on Day 1 of age and nipple counts of male offspring on Day 13 of age showed no adverse effects from parental treatment. 

Male and female offspring body weight and body weight change for F0 males and females receiving 40 mg/kg/day were slightly high when compared with the controls (this could be attributed to to one small litter).

Macroscopic examination of offspring that died prior to the scheduled termination or were killed on Day 13 of gestation did not reveal any findings that were considered related to parental treatment at any dose level.

Reproductive performance and fertility seem to have been affected by treatment at 40 mg/kg bw. There was no adverse effect of treatment on the number of implantations, litter size or the growth of the offspring.

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive toxicity was considered to be 15 mg/kg/day. The NOAEL for developmental toxicity is 40 mg/kg bw.

In the supporting study, carried out on Direct Violet 051, male and female animals administered at 0, 20, 40 and 80 mg/kg bw/day were examined (mating, post-mating, histopathology; the latter in male animals only).

Doses of 0 (vehicle only), 20, 40 and 80 mg/kg bw/day were orally administered (by gavage) to four groups of Han:WIST rats consisting of six animals per group and sex at a dosing volume of 5 mL/kg in concentrations of 4, 8 and 16 mg/mL. A group of vehicle distilled water) treated animals (n= 5/sex) served as a control.

Detailed clinical observations were performed daily after the treatment. On Day 0, animals were observed continuously for 30 minutes then 1, 2, 3 and 5 hours after the administration to estimate the peak period of effects. Body weights were recorded weekly. The food consumption was determined weekly to coincide with body weight measurements during the study. Clinical pathology (hematology, blood coagulation and clinical chemistry) and gross pathology examinations were conducted on all male animals and on female animals of control, 20 and 40 mg/kg bw/day groups one day after the last treatment (on Day 28). Selected organs were weighed.

Male and female animals at 80 mg/kg bw/day were paired form Day 14 until evidence of copulation (positive vaginal smear) was observed. Female animals were observed up to Day 39 (23-25 days after mating) and subjected to necropsy on Day 40.

The results of this study were summarized as follows:

Mortality:There was no mortality in control, 20, 40 or 80 mg/kg bw/daygroups.

Clinical observations:There were no clinical signs in any animals during the treatment period (control, 20, 40 or 80 mg/kg bw/day).The behavior and physical state of animals were normal at the daily and at the detailed weekly clinical observations.

Body weight and body weight gain: The mean body weight gain was slightly depressed in male and female animals at 80 mg/kg bw/day. This slight change resulted in minor changes in the mean body weight of male animals (≤ 8 % of the control) but not in the female animals.

Therefore, test item influence on the body weight development was considered to be of no toxicological relevance.

Food consumption:The mean daily food consumption was not adversely affected by the test item in male or female animals during the entire observation period.

Hematology and blood coagulation:There were no test item related alterations in the examined hematological parameters in male animals at 20, 40 or 80 mg/kg bw/day or in female animals at 20 40 mg/kg bw/day compared to their controls.

Clinical chemistry:Significantly higher mean enzyme activity was observed at 40 and 80 mg/kg bw/day referring to the test item effect as follows: alanine aminotransferase (ALT) in male animals at 80 mg/kg bw/day; aspartate aminotransferase (AST) in male and female animals at 40 mg/kg bw/day and in male animals at 80 mg/kg bw/day).Histopathological examinations could reveal the nature of changes in the liver.

Necropsy:The testes were judged to besmaller than normal in all male animals at 80 mg/kg bw/day in full accordance with organ weight values and histopathological findings.

Organ weight:Test item related reduction of testes weights (absolute and relative to body and brain weights) were detected in male animals at 80 mg/kg bw/day.

Histopathology:Histological investigations revealed decreased intensity of spermatogenesis in the seminiferous tubuli in the testes in moderate or marked degree and lack of spermatozoa in the ductuli of the epididymides in all male animals at 80 mg/kg bw/day.

Reproduction:All female animals showed positive vaginal smear (sign of copulation) and corpora lutea (indicative of fertilization). However, there were no implantation sites in the uterine horns i.e. pre-implantation loss (100 %) occurred in all examined female animals.

Under the conditions of the present study, Direct Violet 51 caused reduced testes weights along with histological lesions (decreased intensity of spermatogenesis in the testes and lack of spermatozoa in the ductuli epididymides) in male Han:WIST rats after the consecutive 28-day oral (by gavage) administration of 80 mg/kg bw/day.

At the same dose level, 100% pre-implantation loss was detected in the female animals.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Quality of whole database:
No effects observed in the OECD 422 study
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

No effects observed at the highest dose tested in the OECD 422 study (NOAEL = 40 mg/kg bw)

Justification for classification or non-classification

Based on the effects on the male reproductive system and the reduced pregnancy rate in females the substance is classified as H361 (Suspected of damaging fertility) according to Regulation (EC) No 1272/2008 (CLP).

Additional information