Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Oct. 04, 1988 to Nov. 01, 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
(Animals were treated during organogenesis Day 5 through 15 of gestation instead of guideline recommendation of dosing through the day of implantation to one day prior to scheduled kill.)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Animals were treated during organogenesis Day 5 through 15 of gestation instead of guideline recommendation of dosing through the day of implantation to one day prior to scheduled kill.
GLP compliance:
yes
Remarks:
according to US FDA and OECD principles of GLP
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 2-Amino-6-chloro-4-nitrophenol, Chlororange
- TSIN: COS 198
- Substance type: Pure active substance
- Physical state: Orange- yellow fine grained powder
- Stability under test conditions: Not reported
- Storage condition of test material: Normal room temperature, in darkness

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Wella AG ; n°batch WS/ COS 198
- Expiration date of the lot/batch: No data
- Purity test date: No data
- Purity : 100 area% (HPLC at 254 nm)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient, 20°C, protected against light
- Stability under test conditions: proven at study initiation over a period of 20 hours by UV-spectroscopy
- Solubility and stability of the test substance in the solvent/vehicle: Yes (the stability was checked on a sample of a low dose level - The results revealed a sufficient stability within 20 hours and concentration values did not exceed a range of +/- 3% from the nominal values.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING No

Test animals

Species:
rat
Strain:
Wistar
Remarks:
derived SPF-Albino rats of the Crl:Wi/Br strain
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga, Versuchstierzucht
- Age at study initiation: Males - 10 weeks old and females - 8 weeks old
- Weight at study initiation: Males: About 300 g; females : 183-231 g
- Fasting period before study: Not reported
- Housing: Single housed, except during mating, in Makrolon cages.
- Bedding: Altromin Laboreinstreu produced from pure soft wood, dried, disdusted and sterilized at 180° C; renewed weekly
- Diet: Ssniff R pelleted diet (Alleindiat fur Ratten) produced by Ssniff Spezialdiaten GmbH;ad libitum
- Water: Tap water from Makrolon drinking bottles; ad libitum
Food diet was analysed periodically and water samples were analysed twice a year.
- Acclimation period: 9 days
Animals used in this test were examined for their health status and only animals in good health were used for the test. Before initiating the study a second clinical examination was carried out to assure that all animals were in the best condition for this investigation.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.5 ± 1.5°C
- Relative humidity: 65 ± 10%
- Air changes: 16 times/hour (Quality: SPF grade)
- Photoperiod: 12 h artificial light (120 flux) from 7 a.m. - 7 p.m.

IN-LIFE DATES: From Oct. 04, 1988 to Nov. 01, 1988

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
(sodium salt; 0.5%)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Appropriate amounts of the test substance were weighed out for each group and a series of suspensions with corresponding concentrations was prepared in sodium-carboxymethylcellulose for each treatment day. All dosing suspension were prepared fresh daily and administered within two hours after preparation. Preparations were protected against light and were permanently stirred on a magnetic stirrer throughout the application routine.

VEHICLE
- Justification for use and choice of vehicle: Not reported
- Concentration in vehicle: 0.1, 0.3 and 0.9 mg/mL for dose levels of 10, 30 and 90 mg/kg bw/day respectively
- Amount of vehicle: 10 mL/kg b w; the volume of the test substance was adapted daily to the weight development of the animals in order to achieve an exact dosage per body weight.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Time points of analysis: Prior to the first treatment day, once throughout the study, and on termination, concentration analyses were performed
Analyses were performed by IBR-Bio Analytical Centre, Hanover and the results and details of analysis were provided in the appendix of the study report. Concentration values did not exceed a range of ± 3% from the nominal values.
Details on mating procedure:
- Impregnation procedure: Co housed
- M/F ratio per cage: 1:2
- Length of cohabitation: Not reported
- Proof of pregnancy: Spermatozoa observed in vaginal smear considered as Day 0 of pregnancy (Vaginal smears were taken in the early morning hours).

Duration of treatment / exposure:
Days 6 to 15 of gestation
Frequency of treatment:
Females were treated once daily in the mid-morning hours.
Duration of test:
Approximately 4 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
90 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 pregnant females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Randomization to groups: Females, exhibiting a positive vaginal smear, were randomly allocated to the test groups by means of a computerized randomization table.
Justification for the choice of the Species: The rat was chosen for this study because of good fertility, high reproduction rates, good response to teratogens and a great number of background information.
Justification for the dosages Selected: Dose levels used in this study were determined and validated by a pre-test. The dosages were chosen with regard to an anticipated maximal dermal resorption rate of 0.3 % in the human which might correspond to a maximal intake of 0.3 mg/kg, when a 3 % use concentration is employed. The used dose levels correspond therefore to a 33, 100 and 300 fold safety factor compared to the maximum human exposure and were thus considered to represent a sufficient safety factor for a risk assessment of the compound.
Justification for the route of Administration: The route of oral administration was chosen to obtain higher resorption rates compared to dermal application.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily over the entire period of the investigation
- Cage side observations checked included: Sensory and motor behavior, hair coat, urine and fecal excretion and condition of body orifices. Also deviations from normal conditions were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily over the entire period of the investigation
During the treatment phase, special reflex examinations (modified to Irwin) were carried out and the potential effects due to the test substance (dose response) were noted.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded at the beginning (Day 0) of the study and at Days 5, 10, 15 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: Measured for day-intervals 0-5, 5-15, 15-20 and for the entire study period (0-20) on a g food basis
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day #: 20; all mated females were sacrificed by CO2-asphyxiation
- Organs examined: In the pregnant females a complete autopsy and a macroscopic examination of the organs were carried out.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of live/dead fetuses, birth position (anterior/posterior), placentae, individual fetus weights and fetus sex (ano - genital distance).
Fetal examinations:
- External examinations: Yes, all fetuses were examined grossly for external alterations
- Soft tissue examinations: Yes, about 1/3 of the fetuses in each litter were fixed in Bouin's solution, frozen for 14 days and subsequently examined by free-hand blade technique for soft tissue alterations.
- Skeletal examinations: Yes, about 2/3 of the fetuses in each litter were fixed in alcohol, stained with Alizarin Red and examined with magnifier for skeletal defects and variations

Statistics:
- One factorial analyses of variance were calculated for growth parameters and reproduction parameters (number of fetuses, implantations, corpora lutea, weights of fetuses, placentae and uteri).
- Group mean values were compared according to the method of "TUKEY".
- Indices were compared with the U-test of Mann-Whitney for significant differences.
Significance levels are noted as follows: p < 0.05 (slightly significant), p < 0.01 (significant), p < 0.001 (highly significant)
Indices:
Absorption rate = (No. of dams with abortions/Total no. of pregnant dams) x 100
Resorption rate = (No. resorptions/No. implantations) x 100
Post-implantation loss index = ((No. implantations - No. fetuses alive)/No. implantations) x 100
Still birth index = (No. dead fetuses/Total no. of fetuses) x 100
Runts index = (No. runts / Total no. of fetuses) x 100
Variation index = (No. variations/ Total no. of fetuses) x 100
Malformation index = (No. malformations / Total no. of fetuses) x 100
Historical control data:
Yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Urines were dose-relataed orange discolored in all dosed females (test compound induced discoloration)
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
High dose females revealed a significantly reduced weight gain rate only during the treatment period
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption measurements revealed a significantly decreased food consumption durng treatment period in high dose group.
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 examined
Description (incidence and severity):
No gross signs of organ alterations were found in maternal body cavities that could be attributed to treatment.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified

Maternal developmental toxicity

Number of abortions:
not examined
Description (incidence and severity):
None of the control or treated animals had evidence of aborting their pregnancies prior to Day 20 and none delivered prematurely.
Pre- and post-implantation loss:
not examined
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Description (incidence and severity):
The mean numbers of resorptions did not reveal significant differences between the test groups.
Dead fetuses:
not examined
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
not examined
Description (incidence and severity):
The mean number of fetuses per dam was comparable between the control and treated groups.
Description (incidence and severity):
LEFT-RIGHT INTRAUTERINE DISTRIBUTION
The mean number of fetuses per dam in the left or right uterine horn was not dose related different among groups.

BIRTH POSITION
No significant differences were determined among groups, concerning the number of fetuses in anterior or posterior positions

PLACENTA WEIGHTS
Mean placenta weights were comparable between the control and dose groups.

WEIGHT OF UTERI
No significant intergroup differences were determined for uteri-weights.

CORPORA LUTEA
Dose related intergroup differences were found, neither in the total number nor in the number of the left and right ovary.


Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
act. ingr.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
LOAEL
Effect level:
>= 90 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: Maternal toxicity

Results (fetuses)

Fetal body weight changes:
not examined
Description (incidence and severity):
The mean fetal bodyweights did not differ significantly between control fetuses and dose group fetuses
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified
Reduction in number of live offspring:
not specified
Changes in sex ratio:
not examined
Description (incidence and severity):
The mean numbers of male and females per group were considered comparable between the control and each treated group.
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
not examined
Description (incidence and severity):
5 mid dose group fetuses and one high dose group fetus were found with head-neck edemas or whole-body edemas (anomalies).

MALFORMATION INDEX: No fetus was found to be malformed out of 274, 270, 262 and 268 fetuses examined in control, 10, 30 and 90 mg/kg groups respectively. Therefore malformation index is 0 % in all the groups tested
Skeletal malformations:
not examined
Description (incidence and severity):
Examinations of the stained fetal skeletons did not reveal signs of treatment related retardation of the ossification processes. The evaluated data showed a comparable development stage for all test groups. Variations (wavy ribs) were found in all test groups at incidences often seen in this strain of rat.
Visceral malformations:
not examined
Description (incidence and severity):
No adverse effect of treatment was indicated from the fetal organ examinations by free-hand serial sectioning.
Other effects:
not examined
Description (incidence and severity):
RUNTS AND RUNTS INDEX
Runts, fetuses with less than 70% of the mean litter weight of each dam, occurred in the control and 2 treatment groups (10 mg/kg b.w. and 30 mg/kg b.w.), but not in the high dose group (90 mg/kg b.w.). Generally the number of runts per group present in this study is within the normal range for this strain of rat.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
90 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Administration of Chlororange (2-amino-6-chloro-4-nitrophenol) to female Crl:Wi/Br strain Wistar rats by oral gavage during gestation Days 5 to 15 at dose levels of 0, 10, 30 and 90 mg/kg bw resulted in a NOAEL of 30 mg/kg bw for maternal toxicity (Significant reduction in body weight gain and mean food consumption).
The NOAEL for developmental toxicity was established at ≥ 90 mg/kg bw, the highest dose administered.

Executive summary:

The developmental toxicity study of Chlororange (2-amino-6-chloro-4-nitrophenol) was conducted following methods comparable to the OECD Guideline 414 (Prenatal Developmental Toxicity Study).

Female Wistar derived SPF-Albino rats of the Crl:Wi/Br strain (Source: Charles River Wiga, Versuchstierzucht) were used in this study. After receipt, animals were acclimated for 9 days and maintained under standard laboratory conditions (temperature: 21.5 ± 1.5°C, relative humidity: 65 ± 10%, air changes: 16 times/hour, photoperiod: 12 hrs dark / 12 hrs light). The animals were individually housed except during the cohabitation period. The animals were fed on Ssniff R pelleted diet and tap water, ad libitum.

Male and female rats were placed together for mating during the cohabitation period. The presence of spermatozoa in a vaginal smear was considered as Day 0 of gestation. Mated females were assigned randomly to the following treatment groups of 20 females each:

Group 1: Vehicle control (0.5% sodium carboxymethylcellulose)

Group2: 10 mg/kg bw/day

Group 3: 30 mg/kg bw/day

Group 4: 90 mg/kg bw/day

Aqueous 0.5% sodium carboxymethylcellulose served as the vehicle. The test substance was administered to mated female rats by oral gavage once daily from Days 5 through 15 of gestation.

The female rats were observed daily for clinical signs, abortions, premature deliveries and deaths. Body weights were recorded on Day 0 and at Days 5, 10, 15 and 20. Food consumption values were measured for day-intervals 0-5, 5-15, and 15-20 and for the entire study period (0-20).

All rats were sacrificed by CO2 asphyxiation on Day 20, and a macroscopic examination was performed. The number of corpora lutea in each ovary was recorded. The uterus of each rat was examined for pregnancy, number and distribution of implantations, live and dead foetuses, early and late resorptions, birth position (anterior/posterior), and placentae.

Each fetus was identified, weighed and examined for sex and gross external alterations. Approximately one-third of the fetuses in each litter were examined for soft tissue alterations and the remaining fetuses in each litter examined for skeletal alterations.

All females showed normal habits and behaviours throughout the study, and no animaldiedprior to scheduled sacrifice. Females of all dose groups had orange discolored urine throughout the application period at dose related intensity. Mean maternal bodyweight gain and mean food consumption was significantly reduced during the treatment phase in the females of the high dose group (90 mg/kg bw).

Gross necropsy did not reveal any organ alterations attributable to treatment.

No significant differences in the mean number of viable fetuses, the male to female fetal sex ratio, total bodyweights, birth position, number of runts, post-implantation losses, implantations, resorptions, uteri weights, placenta weights and corpora lutea between dosage groups and the control group were observed.

External, skeletal and visceral examinations of fetuses revealed minor variations (wavy ribs) at comparable intergroup frequencies with incidence within the spontaneous variation range of this strain of animals. 5 mid dose group fetuses and one high dose group fetus were found with head-neck edemas or whole-body edemas (anomalies) but since the incidence of this finding revealed no dose relation, it is considered to be coincidental.

Based on the above, administration of Chlororange (2-amino-6-chloro-4-nitrophenol) to female Crl:Wi/Br strain Wistar rats by oral gavage during gestation Days 5 to 15 at dose levels of 0, 10, 30 and 90 mg/kg bw resulted in a NOAEL of 30 mg/kg bw for maternal toxicity (significant reduction in body weight gain and mean food consumption).

The NOAEL for developmental toxicity was established at ≥ 90 mg/kg bw, the highest dose administered.

This developmental toxicity study is classified as acceptable and satisfies the OECD Guideline 414 requirement