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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data from handbook or collection of data

Data source

Reference
Reference Type:
review article or handbook
Title:
Toxicological evaluation of certain veterinary drug residues in food
Author:
FAO/WHO Expert Committee on Food Additives (JECFA)
Year:
2004
Bibliographic source:
FAO/WHO Expert Committee on Food Additives (JECFA),2004

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
other: As mentioned below
Principles of method if other than guideline:
The three-generation reproductive toxicity study of test material was performed in Fischer 344 rats.
GLP compliance:
not specified
Limit test:
no
Justification for study design:
No data available

Test material

Constituent 1
Chemical structure
Reference substance name:
[4-[4,4'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride
EC Number:
208-953-6
EC Name:
[4-[4,4'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride
Cas Number:
548-62-9
Molecular formula:
C25H30N3.Cl
IUPAC Name:
(4-(4,4'-Bis(dimethylamino)benzhydrylidene)cyclohexa-2,5-dien-1-ylidene)dimethylammonium, chloride
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report):Gentian violet
- IUPAC name: N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride
- Molecular formula : C25H30N3.Cl
- Molecular weight : 407.986 g/mol
- Smiles notation: C(\c1ccc(N(C)C)cc1)(c1ccc(N(C)C)cc1)=C1\C=C\C(=[N+](/C)C)C=C1.[ClH-]
- InChl:1S/C25H30N3.ClH/c1-26(2)22-13-7-19(8-14-22)25(20-9-15-23(16-10-20)27(3)4)21-11-17-24(18-12-21)28(5)6;/h7-18H,1-6H3;1H/q+1;/p-1
- Substance type: Organic
- Physical state: Solid

Test animals

Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
No data available
Sex:
male/female
Details on test animals or test system and environmental conditions:
No data available

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Feed
Details on exposure:
No data available
Details on mating procedure:
- M/F ratio per cage:No data available
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy:No data available
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.No data available
- Further matings after two unsuccessful attempts: [no / yes (explain)]No data available
- After successful mating each pregnant female was caged (how): single
- Any other deviations from standard protocol:No data available
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data available
Duration of treatment / exposure:
total:280-294
F0 generation :at least 80days
F1b generation:100-140 days
F2b generation:100-140 days
F3a generation:up to weaning
Frequency of treatment:
Daily
Details on study schedule:
- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
After at lest 80 daysfrom start of study, In F0 generation male and female rats of same dose group were caged together for 14 days for mating .Pups from this mating F1a generation used for separate study while F0 generation animals were mating second times in same dose group following birth of F1b generation


- Selection of parents from F1 generation when pups were [...] days of age.:
At 100-140 days of age, one male and one female from each litter were randomly selected to produce F2a generation. Same procedure was used to produce F2b generation

- Age at mating of the mated animals in the study: [...] weeks
After 100-140 days, F3a generation were produce as result of mating one male and one female animal per litter from F2b generation.
Doses / concentrations
Remarks:
0, 5,15 and 30 mg /kg bw /day
No. of animals per sex per dose:
Total :450
Control group:180
Male :90
female 90
treated group:270
5mg/kg bw/day:45male and 45 female
15mg/kg bw/day:45male and 45 female
30mg/kg bw/day:45male and 45 female
Control animals:
yes, concurrent vehicle
Details on study design:
No data available
Positive control:
No data available

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

OTHER:No data
Oestrous cyclicity (parental animals):
No data available
Sperm parameters (parental animals):
No data available
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no]: No data available
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); for F1b and F2a generation one male and one female from each litter were seleted and 2 male and 2 female per litter for F3a generation were selected excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:yes
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, were observed

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]No data available

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:No data available

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:No data available
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]No data available
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.] No data available

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]No data available

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.: yes in F3a generations were observed
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: No data available

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]No data available

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively. yes in F3a generation
Statistics:
No data available
Reproductive indices:
No data available
Offspring viability indices:
No data available

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No dose related effect on mortality was observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The dose related effect in body weight was observed at dose 30mg/kg bw /day group compared to control and lower dose group.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
No effect observed on fertility index and stillborn animals ,sex ratio, number of pups per litter .

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: No effect observed on reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:
not specified
System:
other: not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
not dose related mortality was observed,
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The dose related effect in body weight was observed at dose 30mg/kg bw /day group compared to control and lower dose group
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
No effect observed on fertility index and stillborn animals ,sex ratio, number of pups per litter .

Effect levels (P1)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: No effect observed on reproductive performace

Target system / organ toxicity (P1)

Critical effects observed:
not specified
System:
other: not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No dose related effects on incidence of gross deformities were noted
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
other: F2b
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: gross deformities
Remarks on result:
other: No effect observed at dose 30mg/kg bw

Target system / organ toxicity (F1)

Critical effects observed:
not specified
System:
other: not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Dose related trend for focal dilatation of the renal cortex and tubules ,for necrosis of the thymus and inverse dose-response relationship for red pulp haematopoietic cell proliferation of the spleen
Other effects:
not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not specified

Effect levels (F2)

Dose descriptor:
LOAEL
Generation:
other: F3a
Effect level:
5 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: Dose related effect observed

Target system / organ toxicity (F2)

Critical effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg bw/day (nominal)
Organ:
kidney
spleen
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified
Treatment related:
not specified
Relation to other toxic effects:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL for reproductive toxicity was considered to be 30 mg/kg bw/day for P, P1 and F2b generation and LOAEL was considered to be 5mg/kg bw/day for F3a generation when male and female Fischer 344 rats were treated with test material orally.
Executive summary:

In three generation reproductive toxicity study, Total 450 male and female Fischer 344 rats were used in the study separated in two group i.e. control group and treated group.90 animals of each sex were used in control group. Test material was given in feed at dose concentration 0, 5, 15 and 30 mg /kg bw /day.45 animals of each sex were used for each dose concentration in F0 generation.

 

In F0 generation male and female rats of same dose group were caged together for 14 days for mating .Pups from this mating F1ageneration used for separate study while F0 generation animals were mating second times in same dose group following birth of F1bgeneration. At 100-140 days of age, one male and one female from each litter were randomly selected to produce F2ageneration. Same procedure was used to produce F2bgeneration .After 100-140 days, F3a generation were produce as result of mating one male and one female animal per litter from F2b generation. Pups from all generations were examined for gross deformities while at weaning, 2male and 2 female per litter of F3a generation were randomly selected for histopathology of organs and tissues.

 

 

The dose related effect in body weight was observed at dose 30mg/kg bw /day dose group, as animals in this group had lower body weight compared to control and lower dose group. No dose related effects were observed on reproductive performance as number of pups per litter, fertility index and numbers of stillborn animals were not affected across the generation or dose group.Also No dose related effects were observed on gross deformities in each generation.

 

The only significant histopathological changes noted in F3ageneration were dose related trend for focal dilation of renal cortex and tubules and statistically significant dose related trend for necrosis of thymus (p<0.012 for male and p<0.0001 for female).Also statistically significant inverse dose response relationship for red pulp hematopoietic cell proliferation of spleen (p<0.001 for male and p<0.00001 for female).

 

Hence, NOAEL for reproductive toxicity was considered to be 30mg/kg bw/day for P, P1 and F2b generation and LOAEL w as considered to be 5mg/kg bw/day for F3a generation when male and female Fischer 344 rats were treated with test material orally.