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Description of key information

Reproductivetoxicity study

The data available for 1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates andstructurally similarread across chemicals was reviewed to determine thereproductive toxicity The NOAEL for reproductive toxicity was considered to be 1000 - 2100mg/kg bw for male and 1000-2600 for female as No effects on reproductive parameters were observed .When male and female rats were treated with1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetatesorally. Thus, comparing this value with the criteria of CLP regulation1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates not likely to classify as reproductive toxicant.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data of read across substances
Justification for type of information:
Weight of evidence approach based on structurally similar chemicals
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: As mentioned below
Principles of method if other than guideline:
WoE report is based on three reproductive toxicity studies on rats
1.Reproductive toxicity study of test material in Rats.
2.Toxicology and Carcinogenesis Study of test material in F344 Rats.
3.Combined Repeat Dose and Reproductive / Developmental Toxicity Screening Test of test material by Oral Administration in Rats
GLP compliance:
not specified
Limit test:
no
Justification for study design:
Not specified
Specific details on test material used for the study:
- Name of test material : 1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates
- Molecular formula : C26H34N8O8
- Molecular weight : 586.597 g/mol
- Smiles notation : CC(=O)O{-}.N{+}c1ccc(N=Nc2cccc(N=Nc3ccc(N{+}.O{-}C(C)=O)cc3N{+}.O{-}C(C)=O)c2)c(N{+}.O{-}C(C)=O)c1
- Substance type : Organic
- Physical state : Solid
Species:
rat
Strain:
other: 1.Wistar 2.Fischer 344 3.Crj: CD(SD)
Details on species / strain selection:
Not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
Study 1.
Age: 14 weeks
- Weight at study initiation: 503 g (males); 280 g (females)
Study 2.
- Source: Charles River Japan Co., Ltd
- Age at study initiation: 9 weeks
- Weight at study initiation: 309 to 355 g for males and from 206 to 232 g for females
- Fasting period before study: No data available
- Housing: Rat were housed stainless steel hanging type wire mesh cage, excluded for the period of pregnancy /nursing period. During pregnancy / nursing female were housed in polycarbonate cage with a floor covering for laboratory animals.
- Diet (e.g. ad libitum): Solid feed for experimental animals irradiated with ultraviolet rays after autoclave sterilized nd filtering with a pore size of 5 μm, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
-Temperature (°C): 22 ± 2 ° C.
- Humidity (%):55 ± 15%
- Air changes (per hr): 12 times / hour
- Photoperiod (hrs dark / hrs light): lighting 12 hours / day (7: 00-19: 00)

Route of administration:
other: 1. oral :gavage 2.oral :feed 3.oral : gavage
Vehicle:
other: 1.polyethylene glycol 2.NIH-07 Rat, meal 3.0.5 % Sodium carboxymethylcellulose mixed with 0.1 % Tween 80
Details on exposure:
Study 1.
Details on oral exposure
PREPARATION OF DOSING SOLUTIONS: Test material dissolved in PEG

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test material dissolved in PEG
- Concentration in vehicle: 0, 50, 200 and 1000 mg/kg bw
- Amount of vehicle (if gavage):10ml/kg
- Lot/batch no. (if required):

Study 2.
PREPARATION OF DOSING SOLUTIONS:Test material mixed with NIH-07 Rat meal premix
DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): NIH-07 Rat as meal, - Storage temperature of food:2 weeks for 45 °C

VEHICLE
- Justification for use and choice of vehicle (if other than water):No data
- Concentration in vehicle: No data
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data
Study 3.
VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5 % Sodium carboxymethylcellulose mixed with 0.1 % Tween 80
- Concentration in vehicle: 0 (vehicle), 100, 300, 1000 mg/kg
- Amount of vehicle (if gavage): 10 mL / kg
- Lot/batch no. (if required): No data
- Purity: No data

Details on mating procedure:
Study 1.
- M/F ratio per cage:1:1
- Length of cohabitation:
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy:presence of spermatozoa in a vaginal smear
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how):
- Any other deviations from standard protocol:
Study 3.
- M/F ratio per cage: 1:1
- Length of cohabitation: 14th day
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy Plug formation or sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Study 1.
- Method: spectrophotometrically (accuracy, homogeneity and stability)
- Sampling time: week 1 and week 6
Duration of treatment / exposure:
Study 1.
males 4 weeks; females 6-7 weeks; animals were treated 2 weeks before mating and during mating (1:1) treatment was continued; after successful mating (presence of spermatozoa in a vaginal smear) males were treated until a total treatment time of 28 days; females were dosed during pregnancy, were allowed to litter and dosed during the lactation period until they were scheduled for necropsy (4-6 days of lactation)
Study 2.
2 years
Study 3.
42 to 47 days
Frequency of treatment:
Daily
Details on study schedule:
Not specified
Remarks:
Study 1.
0, 50, 200 and 1000 mg/kg bw
Study 2.
0, 425, 1100 and 2100 mg/kg bw for male and 0, 500, 1300 and 2600 mg/kg/day for female
Study 3.
0, 100, 300 and 1000 mg/kg bw
No. of animals per sex per dose:
Study 1.
Total: 80
0 mg/kg: 10 male and 10 female
50 mg/kg: 10 male and 10 female
200 mg/kg: 10 male and 10 female
1000 mg/kg: 10 male and 10 female
Study 2.
Total: 480
0 mg/kg/day: 60 male
425 mg/kg/day: 60 male
1100 mg/kg/day: 60 male
2100 mg/kg/day: 60 male

500 mg/kg/day: 60 female
1300 mg/kg/day: 60 female
2600 mg/kg/day: 60 female
Study 3.
Total: 96
0 (vehicle) mg/kg: 12 male, 12 female
100 mg/kg: 12 male, 12 female
300 mg/kg: 12 male, 12 female
1000 mg/kg: 12 male, 12 female
Control animals:
yes, concurrent vehicle
Details on study design:
Study 2.
- Dose selection rationale:Because levels of test material as high as 2500 or 50,000 mg/kg in the feed did not adversely affect survival and mean body weights in the 17-day and 13-week studies, nor cause any chemical-related lesions, doses of 0, 425, 1100 and 2100 mg/kg bw for male and 500, 1300 and 2600 mg/kg/day for female were selected for the 2-year studies.
Study 3.
Details on study design:
- Dose selection rationale: Test substance was repeatedly administered orally to SD rats of 3 male and 3 female at 0, 100, 300 and 1000 mg / kg doses for 14 days, and as a result, No effect on in general condition, body weight, food consumption. No clear toxicity change due to administration of the test substance was observed in both organ weight and necropsy.
Based on these findings, the high dose was set at the upper limit of 1000 mg / kg prescribed in the OECD guidelines, three doses of 300 mg / kg for the medium dose and 100 mg / kg for the low dose were set at a tolerance of about 3 .
Positive control:
Not specified
Parental animals: Observations and examinations:
Study 1.
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data
Study 2 &3
Mortality, clinical sign, body weight and feed Consumption were examined.
Oestrous cyclicity (parental animals):
Study 3.
Estrous cyclicity were examined.
Number of corpus luteums and the number of
landings were examined
Sperm parameters (parental animals):
Study 1.
Parameters examined in [all/P/F1/F2] male parental generations:
testis weight, epididymis weight,
Litter observations:
Examination of fetuses: as required by OECD 422
Postmortem examinations (parental animals):
- Macroscopy: accessory sex organs, epididymides, ovaries, testes and all macroscopic lesions for all animals; according to OECD 422 for 5 animals/sex/group
- Microscopy: not performed
Study 3.
Hematology, clinical chemistry, Gross pathology and histopathology were examined.
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:

GROSS NECROPSY: yes
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Statistics:
Study 1.Dunnett’s test, Steel test, Fisher’s exact test
Study 2.Probability of survival was estimated by the product-limit procedure of Kaplan and Meier; Neoplasm Incidences were analyzed by adjusting for intercurrent mortality is the prevalence Dinse and Lagakos. alternative methods of statistical analysis were used for rapidly lethal neoplasms, and the Fisher exact test and the Cochran-Armitage trend test. Nonneoplastic Lesion Incidences were analyzed by the Fisher exact test was used, a procedure based on the overall proportion of affected animals. Organ and body weight data analyzed using parametric multiple comparison procedures of Dunnett and Williams. Hematology and clinical chemistry data were analyzed using nonparametric multiple comparison methods of Dunn and Shirley. Jonckheere's test was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (Williams' or Shirley's test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-response trend (Dunnett's or Dunn's test).
Study 3.
Regarding the weighing data, parametric data was tested for equal variance by the Bartlett method, and one-way ANOVA was performed when variance was equal. Where the variances are not equal and nonparametric data were tested by Kruskal-Wallis. When significant difference was observed between groups, multiple comparisons of Dunnett method or Dunnett type were performed. Pathological findings in counting data were examined by χ 2 for a × b . When significant difference was observed, comparison was made between control group and each test substance administered group by Armitage χ 2 test. Other counting data were tested by Fisher's direct stochastic method. The significance level of each test was 5%. For the data on newborn babies, the average value calculated for each mother was set as a sample unit.
Reproductive indices:
Study 3
birth rate, implantation rate, delivery rate were examined.
Offspring viability indices:
Study 3
Viability on day 0 and 4 were examined.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Study 1.Clinical signs like faeces discolouration was observed in all treated females and male ; incidental animals of all dose groups showed lethargy, hunched posture, laboured respiration, salivation, chromodacryorrhoea, alopecia, scabs and piloerection. Diarrhea and erythema of the anus are attributed to the use of PEG as vehicle.
Study 2.No clinical sign were considered to be treatment related.
Study 3.Red feces exhibiting the same color tone as the test substance administered in all sexes were observed daily from the day following the administration start date.
When treated wtih 1000 mg/kg bw, in 2 male loose stools were observed transiently on 22 days after the start of administration, but abnormalities in general conditions considered to be toxic changes.
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Study 1.Male : 1/10 at untreated, 50 and 200 mg/kg bw,No mortality in females was observed
Study 2.When treated with 2100 and 2600 mg/kg bw, Significant increase in survival of treated male and female rats were observed as compared to control. When treated with 1100 mg/kg bw, Significant increase in survival of treated male rats were observed as compared to control.
The greater survival of the exposed groups was due principally to the chemically related decreased incidence of mononuclear cell leukemia.
Study 3.No effect on survival of treated male and female rat were observed as compared to control.

Body weight and weight changes:
no effects observed
Description (incidence and severity):
Study 2.When treated with 1300 and 2600 mg/kg bw, Significant decrase in body weights of treated female rats were observed as compared to control.
Study 3.No significant effect on body weight and body weight gain of treated male and female rats were observed as compared to control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Study 2& 3.No effect on food consumption of treated male and female rats was observed as compared to control.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Study 1. Male : RBC increased at 50 mg/kg ,Female : RBC, Hb/haematocrit increased at 50 mg/kg
Study 2.When treated with 2600 mg/kg bw in female, significant decrase in Hematocrit values, hemoglobin concentration, and erythrocyte counts were observed as compared to control. Which indicat mild anemia.
No effect in mlae rats were observed as compared to control.
Study 3.When treated wtih 100 mg / kg, significant decrease in prothrombin time were observed in male rats, but no change was observed in the 300 and 1000 mg / kg group, so it was judged to be an accidental change.
In females, there was no significant difference between the test substance administered group and the control group.


Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Study 1.
male:decreased ALAT/ASAT and increased at 200 mg/kg
Female :ALAT/ASAT increase at 1000 mg/kg; phosphate decreased and glucose increased at 200 mg/kg; creatinine decreased at 50 mg/kg
* The increased liver enzymes in high dosed females can be attributed to a single female. Excluding this female leads to mean group values for ASAT and ALAT that are comparable with control values
Effects on blood parameters showed no relationship with the dose and were observed in one of the sexes only. Therefore they were considered to be not toxicological relevant.
Study 2.
When treated with 2600 mg/kg bw in female, Significant increase in Serum total bilirubin were observed as compared to control.
This finding coupled with the mild anemia suggests a mild hemolytic process.
No effect in mlae rats were observed as compared to control.
Study 3.When treated wtih 1000 mg / kg, significant incrase in A / G ratio were observed in male rats. However, since the total protein and albumin were not changed and were slightly high, it was considered to be fluctuating within the physiological range and it was judged that it was not caused by administration of the test substance.

When treated wtih 100 mg / kg, significant incrase in ASAT (GOT) in female rats, but no change was observed in the 300 and 1000 mg / kg group, so it was judged to be an accidental change .
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Study 2.When treated with 2100 mg/kg bw in male rats, Four renal tubule cell adenomas or carcinomas, renal tubule focal hyperplasia and renal tubule adenomas were observed.When treated with 1100 and 2100 mg/kg bw in male rats, Four renal tubule cell adenomas or carcinomas were observed.When treated with 2500 mg/kg bw in female rats, one renal tubule adenoma was observed.
Although the trend for these renal neoplasms is significant, the incidences are low and do not exceed the historical control range of 0% to 6% in male rats. When treated with 2100 and 2600 mg/kg bw in male and 1300 and 2600 mg/kg bw in female, significant dose-related decrease in the incidence of mononuclear cell leukemia was observed as compared to control. No significant histopathological changes were observed in reproductive organ such as testes and mammory gland of treated male and female rats as compared to control.
Study 3.
No histopathological changes were observed in treated male and female rats as compared to control.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Study 3.No effect on sexual cycle of treated female rat were observed as compared to control.
Reproductive performance:
no effects observed
Description (incidence and severity):
Study 1.
No effects on weight and no gross findings in the reproductive organs.). No additional findings on the testes were present in any of these animals. The effect on the preputial gland is a common finding in young male rats. No relationship to treatment is suspected.
- Successful mating: no treatment related effects
- % mated: 100%
- Number pregnant per dose level: 9/10 for all dose levels
- Number aborting: none
- Number of implantations: no treatment related effects
- Duration of gestation: 21-22 days

Study 3.No significant difference was observed in mating index, fertility index, numbers of corpora lutea or implantations, implantation index, delivery index, gestation index, gestation length, parturition or maternal behavior, numbers of offspring or live offspring of treated rats as compared to control.

Dose descriptor:
NOAEL
Effect level:
> 1 000 - <= 2 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive performance
Remarks on result:
other: overall no toxic effetcs was observed
Dose descriptor:
NOAEL
Effect level:
> 1 000 - <= 2 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
reproductive performance
Remarks on result:
other: No effects on reproductive performance
Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
Study 3.
No abnormal clinical signs were observed in offspring.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Study 1.
Viability: Number of litters: 9 at all doses
- Number of litters: 9 at all doses
- Number of dead pups/no litters: at 0, 50, 200 and 1000 mg/kg bw 3/2, 3/2, 1/1 and 0/0, respectively
- Mean live pups/litter: 0, 50, 200 and 1000 mg/kg bw 13, 12, 15 and 14, respectively
- Postnatal loss/no litters: 0, 50, 200 and 1000 mg/kg bw 5/2, 17/4, 9/3 and 6/5, respectively

*. At 50 mg/kg bw 13 pups were lost in one litter
Study 3.
No effect on numbers of live offspring of were observed as compared to control.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Study 3.No significant change in body weights of offspring were observe as compared to control.
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:
effects observed, non-treatment-related
Description (incidence and severity):
Study 1.
Macroscopy pups: at 200 mg/kg one pup with malformations was found (a.o. open back/skull); yellow skin in 2 of 10 litters at 1000 mg/kg.The discolourations observed in parents and pups are considered to be related to the staining effect of test substance. From their location it can be concluded that the test substance did not become available systemically, which is in line with other publications on Diarylide Yellow Pigments
Study 3.
No gross pathological changes were observed in offsprings.
Histopathological findings:
not specified
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
viability
mortality
body weight and weight gain
gross pathology
Remarks on result:
other: overall no effects on developmental parameters
Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified
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
Conclusions:
NOAEL was considered to be 1000 - 2100mg/kg bw for male and 1000-2600 for female ,when rats were treated with 1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates orally .
Executive summary:

Data available from different studies were reviewed to determine the reproductive toxicity of 1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates .The studies are as mentioned below:

Study 1.

In a reproductive toxicity study, male and female wistar rats were treated with test material in the concentration of 0, 50, 200 and 1000 mg/kg bw orally by gavage formales 4 weeks; females 6-7 weeks; animals were treated 2 weeks before mating and during mating (1:1) treatment was continued; after successful mating (presence of spermatozoa in a vaginal smear) males were treated until a total treatment time of 28 days; females were dosed during pregnancy, were allowed to litter and dosed during the lactation period until they were scheduled for necropsy (4-6 days of lactation). The test material dissolved in PEG and analyzed by spectrophotometrically (accuracy, homogeneity and stability).10 animals/sex /dose group were used. All the animals were observed for Clinical signs, Body weight gain, Food consumption and Examination of uterine content and fetuses were carried out. Macroscopic examination of accessory sex organs, epididymides, ovaries, testes and all macroscopic lesions for all animals; according to OECD 422 for 5 animals/sex/group were done were as Microscopy not performed.

No mortality in females was observed while in male 1/10 at untreated, 50 and 200 mg/kg bw

Were observed. Clinical signs like faeces discolouration was observed in all treated females and male ; incidental animals of all dose groups showed lethargy, hunched posture, laboured respiration, salivation, chromodacryorrhoea, alopecia, scabs and piloerection. Diarrhea and erythema of the anus are attributed to the use of PEG as vehicle. No treatment related effects onBody weight, food consumption, organweights andhistopathology. Gross pathology in female showed 1/10 greenish contents of the caecum at 1000 mg/kg

 

 In male hematological examination showed RBC increased at 50 mg/kg while decreased ALAT/ASAT and increased at 200 mg/kg.In female hematological examination showed RBC, Hb/hematocrit increased at 50 mg/kg while ALAT/ASAT increase at 1000 mg/kg; phosphate decreased and glucose increased at 200 mg/kg; creatinine decreased at 50 mg/kg. Effects on blood parameters showed no relationship with the dose and were observed in one of the sexes only. Therefore they were considered to be not toxicological relevant,

The increased liver enzymes in high dosed females can be attributed to a single female. Excluding this female leads to mean group values for ASAT and ALAT that are comparable with control values. The inflammation of the preputial glands seen in 4 of 5 high dosed males was minimal (grade 1-2). No additional findings on the testes were present in any of these animals. The effect on the preputial gland is a common finding in young male rats. No relationship to treatment is suspected

No effects on weight and no gross findings in the reproductive organs. Successful mating as no treatment related effects was observed. Number pregnant per dose level: 9/10 for all dose levels. No abortion was observed. No treatment related effects on Number of implantations and duration of gestation. No effects onViability,Number of dead pups/no litters and Mean live pups/litter was observed. Body weight/sex pups were unchanged.
In macroscopic examination of pups at 200 mg/kg one pup with malformations was found (a.o. open back/skull); yellow skin in 2 of 10 litters at 1000 mg/kg was observed. The discolourations observed in parents and pups are considered to be related to the staining effect of test substance. From their location it can be concluded that the test substance did not become available systemically, which is in line with other publications on Diarylide Yellow Pigments. Hence
NOAEL was considered to be 1000 mg/kg bw for P and F1 generation whenwistar rats were treated with test material orally by gavage according to OECD guideline 422.

 

Study 2.

In a Toxicology and Carcinogenesis Study, F344 male and female rat were treated with test material in the concentration of 0,425, 1100 and 2100 mg/kg bw for male and 0, 500, 1300 and 2600 mg/kg/day for female orally in diet for 2 years. The average daily ingestion of test material was approximately 425, 1,100, or 2,100 mg/kg body weight per day for male rats and 500, 1,300, or 2,600 mg/kg for females. Significant increase in survival of treated male and female rats were observed at 2100 and 2600 mg/kg bw as compared to control. Significant increase in survival of treated male rats were observed at 1100 mg/kg bw as compared to control. The greater survival of the exposed groups was due principally to the chemically related decreased incidence of mononuclear cell leukemia. No clinical sign were considered to be treatment related. Significant decrease in body weights of female rats were observed at 1300 and 2600 mg/kg bw as compared to control.No effect on food consumption of treated male and female rats was observed as compared to control.In female, significant decrease in Hematocrit values, hemoglobin concentration, and erythrocyte counts at 2600 mg/kg bw as compared to control. Which indicate mild anemia. No effect in male rats was observed as compared to control. Significant increase in Serum total bilirubin were observed in female rats at 2600 mg/kg bw as compared to control. This finding coupled with the mild anemia suggests a mild hemolytic process. No effect in male rats was observed as compared to control. Similarly, at 1300 and 2600 mg/kg bw in female rats, significant increase in relative kidney and brain weight were observed. In addition, at 2100 mg/kg bw in male rats, Four renal tubule cell adenomas or carcinomas, renal tubule focal hyperplasia and renal tubule adenomas were observed. At 1100 and 2100 mg/kg bw in male rats, Four renal tubule cell adenomas or carcinomas were observed. At 2500 mg/kg bw in female rats, one renal tubule adenoma was observed. Although the trend for these renal neoplasms is significant, the incidences are low and do not exceed the historical control range of 0% to 6% in male rats. At 2100 and 2600 mg/kg bw in male and 1300 and 2600 mg/kg bw in female,significant dose-related decrease in the incidence of mononuclear cell leukemia was observed as compared to control.No significant histopathological changes were observed in reproductive organ such as testes and mammary gland of treated male and female rats as compared to control.Therefore, NOAEL was considered to be 500 mg/kg/day for P and F1 generation whenWistarmale and female rats treated withIndigo Carmineorally in dietfor 2 years. Therefore,NOAEL was considered to be 2100 mg/kg/day for male and 2600 mg/kg bw for female P generation whenF344male and female rats treated with test material  orally in diet for 2 years.

Study 3

In a Combined Repeat Dose and Reproductive / Developmental Toxicity Screening Test, Crj:CD(SD) male and female rats treated with test material in the concentration of 0 (vehicle), 100, 300, 1000 mg/kg orally by gavage for 42 to 47 days. No effect on survival of treated male and female rat was observed as compared to control. Red feces exhibiting the same color tone as the test substance administered in all sexes were observed daily from the day following the administration start date. At 1000 mg/kg bw, in 2 male loose stools were observed transiently on 22 days after the start of administration, but abnormalities in general conditions considered to be toxic changes. No significant effect on body weight and body weight gain and food consumption of treated male and female rats were observed as compared to control. Significant decrease in prothrombin time were observed in male rats at 100 mg / kg/day, but no change was observed in the 300 and 1000 mg / kg group, so it was judged to be an accidental change. In females, there was no significant difference between the test substances administered group and the control group. Significant increase in A / G ratio were observed in male rats. At 1000 mg / kg/day However, since the total protein and albumin were not changed and were slightly high, it was considered to be fluctuating within the physiological range and it was judged that it was not caused by administration of the test substance. Significant increase in ASAT (GOT) in female rats at 100 mg / kg/day, but no change was observed in the 300 and 1000 mg / kg group, so it was judged to be an accidental change. Similarly, No effect on reproductive parameters such as sexual cycle, mating index, the fertility index, numbers of corpora lutea or implantations, the implantation index, the delivery index, the gestation index, gestation length, parturition or maternal behavior, numbers of offspring or live offspring of treated rats as compared to control. Significant increase in relative liver weight in male and absolute and relative weight in female were observed at 1000 mg / kg bw as compared to control. Significant decrease in absolute spleen weight and significant increase in relative adrenal gland weight were observed at 300 mg / kg bw in male rats. Significant increase in relative adrenal gland weight were observed in male rats at 100 mg / kg bw. But, since there was no change in the 1000 mg / kg group, it was judged to be an accidental change. Colored stomach at 1000 mg / kg and red, colored contents were observed in small intestine and large intestine at 100 mg / kg. Focal bleeding of the lung, contraction failure and white spots, stomach tar-like contents, white spots of the liver, dilation of the renal pelvic cavity, uterine bloating, vaginal distension and ventricular dilation were observed. However, since there was no certain tendency in its expression status, it was judged to be an accidental change unrelated to administration of the test substance. No histopathological changes were observed in treated male and female rats as compared to control. In addition, No effect on numbers of live offspring, clinical signs and body weight of offspring were observed as compared to control. No gross pathological changes were observed in offspring. Therefore, NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Crj:CD (SD) male and female rats treated with test material orally by gavage for 42 to 47 days.

 Based on the data available from different studies, 1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates did not showedreproductive toxicityat dose concentration1000mg/kg bw/day.Hence the test chemical is not likely to classify as a reproductive toxicant as per the criteria mentioned in CLP regulation.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Data is Klimicsh 2 and from authoritative database
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproductive toxicity study

Data available from different studies were reviewed to determine the reproductive toxicity of 1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates .The studies are as mentioned below:

Study 1.

In areproductive toxicity study, male andfemale wistar rats were treated with test materialin the concentration of 0, 50, 200 and 1000 mg/kg bworally by gavage formales 4 weeks; females 6-7 weeks; animals were treated 2 weeks before mating and during mating (1:1) treatment was continued; after successful mating (presence of spermatozoa in a vaginal smear) males were treated until a total treatment time of 28 days; females were dosed during pregnancy, were allowed to litter and dosed during the lactation period until they were scheduled for necropsy (4-6 days of lactation). The test material dissolved in PEG and analyzed by spectrophotometrically (accuracy, homogeneity and stability).10 animals/sex /dose group were used. All the animals were observed for Clinical signs, Body weight gain, Food consumption and Examination of uterine content and fetuses were carried out. Macroscopic examination of accessory sex organs, epididymides, ovaries, testes and all macroscopic lesions for all animals; according to OECD 422 for 5 animals/sex/group were done were as Microscopy not performed.No mortality in females was observed while in male 1/10 at untreated, 50 and 200 mg/kg bw

Were observed. Clinical signs like faeces discolouration was observed in all treated females and male ; incidental animals of all dose groups showed lethargy, hunched posture, laboured respiration, salivation, chromodacryorrhoea, alopecia, scabs and piloerection. Diarrhea and erythema of the anus are attributed to the use of PEG as vehicle. No treatment related effects onBody weight, food consumption, organweights andhistopathology. Gross pathology in female showed 1/10 greenish contents of the caecum at 1000 mg/kgIn male hematological examination showed RBC increased at 50 mg/kg while decreased ALAT/ASAT and increased at 200 mg/kg.In female hematological examination showed RBC, Hb/hematocrit increased at 50 mg/kg while ALAT/ASAT increase at 1000 mg/kg; phosphate decreased and glucose increased at 200 mg/kg; creatinine decreased at 50 mg/kg. Effects on blood parameters showed no relationship with the dose and were observed in one of the sexes only. Therefore they were considered to be not toxicological relevant,

The increased liver enzymes in high dosed females can be attributed to a single female. Excluding this female leads to mean group values for ASAT and ALAT that are comparable with control values. The inflammation of the preputial glands seen in 4 of 5 high dosed males was minimal (grade 1-2). No additional findings on the testes were present in any of these animals. The effect on the preputial gland is a common finding in young male rats. No relationship to treatment is suspected

No effects on weight and no gross findings in the reproductive organs. Successful mating as no treatment related effects was observed. Number pregnant per dose level: 9/10 for all dose levels. No abortion was observed. No treatment related effects on Number of implantations and duration of gestation. No effects onViability,Number of dead pups/no litters and Mean live pups/litter was observed. Body weight/sex pups were unchanged.
In macroscopic examination of pups at 200 mg/kg one pup with malformations was found (a.o. open back/skull); yellow skin in 2 of 10 litters at 1000 mg/kg was observed. The discolourations observed in parents and pups are considered to be related to the staining effect of test substance. From their location it can be concluded that the test substance did not become available systemically, which is in line with other publications on Diarylide Yellow Pigments. Hence
NOAEL was considered to be 1000 mg/kg bw for P and F1 generation whenwistar rats were treated with test materialorally by gavageaccording to OECD guideline 422.

 

Study 2.

In a Toxicology and Carcinogenesis Study, F344 male and female rat were treated with test material in the concentration of 0,425, 1100 and 2100 mg/kg bw for male and 0, 500, 1300 and 2600 mg/kg/day for female orally in diet for 2 years. The average daily ingestion of test material was approximately 425, 1,100, or 2,100 mg/kg body weight per day for male rats and 500, 1,300, or 2,600 mg/kg for females. Significant increase in survival of treated male and female rats were observed at 2100 and 2600 mg/kg bw as compared to control. Significant increase in survival of treated male rats were observed at 1100 mg/kg bw as compared to control. The greater survival of the exposed groups was due principally to the chemically related decreased incidence of mononuclear cell leukemia. No clinical sign were considered to be treatment related. Significant decrease in body weights of female rats were observed at 1300 and 2600 mg/kg bw as compared to control.No effect on food consumption of treated male and female rats was observed as compared to control.In female, significant decrease in Hematocrit values, hemoglobin concentration, and erythrocyte counts at 2600 mg/kg bw as compared to control. Which indicate mild anemia. No effect in male rats was observed as compared to control. Significant increase in Serum total bilirubin were observed in female rats at 2600 mg/kg bw as compared to control. This finding coupled with the mild anemia suggests a mild hemolytic process. No effect in male rats was observed as compared to control. Similarly, at 1300 and 2600 mg/kg bw in female rats, significant increase in relative kidney and brain weight were observed. In addition, at 2100 mg/kg bw in male rats, Four renal tubule cell adenomas or carcinomas, renal tubule focal hyperplasia and renal tubule adenomas were observed. At 1100 and 2100 mg/kg bw in male rats, Four renal tubule cell adenomas or carcinomas were observed. At 2500 mg/kg bw in female rats, one renal tubule adenoma was observed. Although the trend for these renal neoplasms is significant, the incidences are low and do not exceed the historical control range of 0% to 6% in male rats. At 2100 and 2600 mg/kg bw in male and 1300 and 2600 mg/kg bw in female,significant dose-related decrease in the incidence of mononuclear cell leukemia was observed as compared to control.No significant histopathological changes were observed in reproductive organ such as testes and mammary gland of treated male and female rats as compared to control.Therefore, NOAEL was considered to be 500 mg/kg/day for P and F1 generation whenWistarmale and female rats treated withIndigo Carmineorally in dietfor 2 years. Therefore,NOAEL was considered to be 2100 mg/kg/day for male and 2600 mg/kg bw for female P generation whenF344male and female rats treated with test material  orally in diet for 2 years.

Study 3

In a Combined Repeat Dose and Reproductive / Developmental Toxicity Screening Test, Crj:CD(SD) male and female rats treated with test material in the concentration of 0 (vehicle), 100, 300, 1000 mg/kg orally by gavage for 42 to 47 days. No effect on survival of treated male and female rat was observed as compared to control. Red feces exhibiting the same color tone as the test substance administered in all sexes were observed daily from the day following the administration start date. At 1000 mg/kg bw, in 2 male loose stools were observed transiently on 22 days after the start of administration, but abnormalities in general conditions considered to be toxic changes. No significant effect on body weight and body weight gain and food consumption of treated male and female rats were observed as compared to control. Significant decrease in prothrombin time were observed in male rats at 100 mg / kg/day, but no change was observed in the 300 and 1000 mg / kg group, so it was judged to be an accidental change. In females, there was no significant difference between the test substances administered group and the control group. Significant increase in A / G ratio were observed in male rats. At 1000 mg / kg/day However, since the total protein and albumin were not changed and were slightly high, it was considered to be fluctuating within the physiological range and it was judged that it was not caused by administration of the test substance. Significant increase in ASAT (GOT) in female rats at 100 mg / kg/day, but no change was observed in the 300 and 1000 mg / kg group, so it was judged to be an accidental change. Similarly, No effect on reproductive parameters such as sexual cycle, mating index, the fertility index, numbers of corpora lutea or implantations, the implantation index, the delivery index, the gestation index, gestation length, parturition or maternal behavior, numbers of offspring or live offspring of treated rats as compared to control. Significant increase in relative liver weight in male and absolute and relative weight in female were observed at 1000 mg / kg bw as compared to control. Significant decrease in absolute spleen weight and significant increase in relative adrenal gland weight were observed at 300 mg / kg bw in male rats. Significant increase in relative adrenal gland weight were observed in male rats at 100 mg / kg bw. But, since there was no change in the 1000 mg / kg group, it was judged to be an accidental change. Colored stomach at 1000 mg / kg and red, colored contents were observed in small intestine and large intestine at 100 mg / kg. Focal bleeding of the lung, contraction failure and white spots, stomach tar-like contents, white spots of the liver, dilation of the renal pelvic cavity, uterine bloating, vaginal distension and ventricular dilation were observed. However, since there was no certain tendency in its expression status, it was judged to be an accidental change unrelated to administration of the test substance. No histopathological changes were observed in treated male and female rats as compared to control. In addition, No effect on numbers of live offspring, clinical signs and body weight of offspring were observed as compared to control. No gross pathological changes were observed in offspring. Therefore, NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Crj:CD (SD) male and female rats treated with test material orally by gavage for 42 to 47 days.

 Based on the data available from different studies, 1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates did not showedreproductive toxicityat dose concentration1000mg/kg bw/day.Hence the test chemical is not likely to classify as a reproductive toxicant as per the criteria mentioned in CLP regulation.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Thus, comparing this value with the criteria of CLP regulation1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates not likely to classify as reproductive toxicant.

Additional information

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