Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Mar. 22, 2004 to Aug. 10, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted : 22nd January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Remarks:
according to Swiss ordinance based on OECD principles of GLP
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material 2-Amino-4-hydroxyethylaminoanisole sulfate (Code# A000157)
- TSIN: WR 23081
- Substance type: Pure active substance
- Physical state: Pale grey powder
- Storage under test conditions: Storage in a tightly closed container in a dry, cool and well-ventilated place protected from humid air, water, heat and light.
- Stability in solution: Small degradation (retrieval rate was between 100 and 79.7% during the storage)of the test substance (approximately 5% in solution)was observed in DMSO and in aqueous solution over a period of 7 days when examined by means of HPLC-UV
- Solubility: 10 g/L in water pH 2.8
1 weight% in acetone/water 1:1 (pH 2.1)
9-10 weight% in DMSO
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Wella, Germany; Batch# Ch. 57/01 (R96000195; R96000196)
- Expiration date of the lot/batch: July 2005
- Purity test date: Feb. 19, 2001

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: kept tightly closed in a dry, cool and well-ventilated place. Protect from humid air and water as from heat and direct sunlight
- Solubility and stability of the test substance in the solvent/vehicle: Small degradation (retrieval rate was between 100 and 79.7% during the storage)of the test substance (approximately 5% in solution)was observed in DMSO and in aqueous solution over a period of 7 days when examined by means of HPLC-UV

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was weighed into a glass beaker and the vehicle added (w/v). The mixtures were prepared using a homogenizer. During the daily administration period, homogeneity was maintained using a magnetic stirrer.
- Final dilution of a dissolved solid, stock liquid or gel: 1 mg/ml (dose group 2 10 mg/kg bw/day), 3 mg/ml (dose group 3, 30 mg/kg bw/day) and 15 mg/ml (dose group 4 150 mg/kg bw/day)

Test animals

Species:
rat
Strain:
Wistar
Remarks:
HanBrl: WIST (SPF)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Laboratory Animals Services, Wolferstrasse 4, CH-4414 Fullinsdorf, Switzerland
- Age at study initiation: 10 weeks minimum
- Weight at study initiation: 189-257 g
- Fasting period before study: Not reported
- Housing: Individually (except during mating) in Makrolon cages (type-3) with wire mesh tops and standardized granulated softwood bedding (Lignocel, Schill AG, CH-4132 Muttenz, Switzerland).
- Diet: Pelleted standard Kilba-Nafag 3433 rat/mouse maintenance diet (Provimi Kilba AG, CH-4303 Kaiseraugst, Switzerland; Batch No. 78/03); ad libitum
- Water: Community tap water from Fullinsdorf in bottles; ad libitum.
- Acclimation period: 10 days prior to mating with an evaluation of the health status.

ENVIRONMENTAL CONDITIONS
- Temperature: Target range 22 ±3°C
- Relative humidity: Target range 30-70%
- Air changes: Air-conditioned with 10-15 air changes per hour
- Photoperiod: 12 hours artificial fluorescent light/12 hours dark with background music played at a centrally defined low volume for at least 8 hours during the light period.

IN-LIFE DATES: From: Mar. 29, 2004 To: Apr. 16, 2004

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(bi-distilled)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Doses were formulated daily. Vehicle was added into a glass beaker containing weighed test substance and mixed with a homogenizer. Homogeneity was maintained during the daily administration period using a magnetic stirrer.

VEHICLE: Bi-distilled water
- Amount of vehicle: 10 mL/kg body weight with a daily adjustment to the actual body weight

ADMISTRATION OF TEST SUBSTANCE: 10 mL/kg bw of test substance was administered daily by oral gavage from Day 6 (first treatment) to Day 20 (last treatment). Control animals were similarly dosed with the vehicle (bi-distilled water) alone. The following treatment groups were employed.
Group 1: Vehicle control (bi-distilled water)
Group2: 10 mg/kg bw/day (low dose)
Group 3: 30 mg/kg bw/day (mid dose)
Group 4: 150 mg/kg bw/day (highdose)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SAMPLING: During the first and last week of the administration period samples for determination of concentration, homogeneity and stability (first week only) were taken. The samples of approximately 2 g were transferred into flat bottomed flasks and frozen (-25°C to -15°C) pending analyses.
- Homogeneity: For determination of homogeneity, samples were taken from the top, middle and bottom of each formulation. The mean values from these analyses also represent the concentration and stability immediately after preparation.
- Stability: Samples for determination of stability were taken after 4 hours storage at room temperature (22 ±3°C).
Samples were sent with dry ice to Dr. D. Flade, RCC Ltd, Environmental Chemistry & Pharmanalytics, CH-4452 Itingen/Switzerland. Analysis was performed using an external standard method provided by the Sponsor (HPLC).
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused:- M/F ratio per cage: 1:1 (one male:one female)
- Length of cohabitation: Until evidence of copulation was observed
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: If either the daily vaginal smear was sperm positive or copulation plug was observed and was designated as Day 0 post coitum.
The females were housed with the males in special automatic mating cages, i.e. with synchronized timing to initiate the right mating period. This system reduced the variation in the copulation times of the different females.
Duration of treatment / exposure:
From Day 6 (implantation) to Day 20 post coitum (day before sacrifice).
Frequency of treatment:
Daily
Duration of test:
3 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for the choice of the Species: Rat is a suitable rodent species for development toxicity studies required by regulatory authorities
- Dose selection rationale: Dose levels were selected in conjunction with the Sponsor, based on the results of a previous dose range-finding study in pregnant rats (RCC study number 851838). In this study, animals were treated with 0, 10, 30, and 90 mg/kg from Day 6 to Day 20 of pregnancy. Slightly reduced food consumption and body weight gain were noted. Therefore the highest dosage for the main study was increased to 150 mg/kg body weight/day. There was no teratogenic effect.
- Rationale for animal assignment: Mated rats were assigned to the different groups using a computer-generated random algorithm.

Examinations

Maternal examinations:
MORTALITY/VIABILITY: Yes
- Time schedule: Animals were checked at least twice daily for mortalities, morbidity or signs of abortion. Animals found dead were necropsied.
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observed at least twice daily for clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from Day 0 until Day 21 post coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Time schedule: Recorded on 3-day intervals: Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum.
- 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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day # 21 post coitum just prior to expected delivery, females were killed by CO2 and the fetuses were removed by Cesarean section
- Organs examined: The main organs of the thoracic and abdominal cavities, in particular the genitals were examined.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (by ammonium sulfide staining)
- Number of corpora lutea: Yes
- Number of implantations: Yes (number and location of live and dead fetuses)
- Number and location of early resorptions: Yes
- Number and location of late resorptions: Yes
- Other: uteri that appeared non-gravid were further examined (e.g. by ammonium sulfide staining) to confirm the non-pregnant status.
Fetal examinations:
- External examinations: Yes, all live fetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, killed by subcutaneous injection of sodium pentobarbital (Vetanarcol) in the scruff and allocated to either visceral or skeletal evaluation. In the case of gross external malformation, fetuses were allocated to the alternate technique depending on the type of finding.
- Soft tissue examinations: Yes, half per litter (using a computer-generated random program at an approximate 1:1 ratio within each litter independent of sex).
Fetal visceral examination/microdissection technique: Fetuses were fixed in Bouin's solution (one fetus per container) for at least two weeks and were microdissected. Serial sections of the head, microdissection of the thorax and abdomen and detailed examination of the major blood vessels were done. After examination, organs and sections were preserved in a solution of glycerin/ethanol (one fetus per container). Observations of visceral abnormalities and variations were recorded.
- Skeletal examinations: Yes: half per litter (using a computer-generated random program at an approximate 1:1 ratio within each litter independent of sex). Fetuses were eviscerated and with the exception of the paws, the skin was removed and discarded. Carcasses were processed through solutions of ethanol, glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alzarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The skeletons were examined and all abnormal findings and variations recorded. The specimens were preserved individually. Fetuses with abnormalities were photographed where applicable.
- Head examinations: No
Statistics:
The following statistical methods were used to analyze body weights, food consumption, reproduction and skeletal examination data:
-Means and standard deviations of various data were calculated and included in the report.
-If the variables could be assumed to follow a normal distribution, the Dunnett many-one t-test, based on a pooled variance estimate, was used for intergroup comparisons (i.e. single treatment groups against the control group).
-The Steel test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
-Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information. All methods of analysis and the results are included in the report.
Indices:
Not reported
Historical control data:
Not reported

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In mid and high dose groups, dark discoloration of urine and bedding were observed for all females from Day 6 post coitum onwards. The discolorations were considered to be due to the presence of test substance (and/or metabolites) and were not considered to be an adverse effect. In low dose group, localized alopecia was transiently noted for one female. This isolated and common finding was considered to be incidental
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No death occurred which was considered to be test substance related. In the 150 mg/kg-bw/day dose group, ruffled fur, incrusted nose and poor condition were noted for one female on days 7 to 9 post coitum. This female was found dead in the morning of Day 10 post coitum. In all probability the death of this female was the consequence of an injury caused by intubation on the first day of test substance administration (Day 6 post coitum). All other females survived until scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group, mean body weight gain was slightly reduced only up to Day 16 post coitum Further, the corrected mean body weight gain (corrected for gravid uterus weight) was slightly lower (+3.4% vs. +7.4% in the vehicle control).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
: In the high dose group, reduced mean food consumption was noted during the entire treatment period (7.4%).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
During macroscopic examination no test item-related findings were noted. In group 3, the non-gravid uterus of one female (No. 49) contained fluid, which is a common finding in non-pregnant rats of this strain. No abnormal findings were noted in females of group 4, 2 or vehicle controls.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of pre-implantation loss was statistically significantly lower in the 30 and 150 mg/kg-bw/day dose groups compared to the vehicle control resulting in a slightly higher number of implantations in this group. Since pre-implantation loss would have occurred primarily prior to the onset of treatment this effects is considered to be incidental. Statistically significant differences noted for post-implantation loss, embryonic and fetal resorptions in low dose group were due to a lower incidence of post-implantation loss which resulted in a statistically significantly higher value of fetuses as a percentage of implantation sites. The same applies for the statistically significant lower incidence of fetal resorptions noted in the 30 mg/kg-bw/day dose group. In the 90 mg/kg-bw/day dose group, the incidence of embryonic resorptions was slightly although statistically significantly increased, which was considered to be likely due to the high number of implantation sites per dam (13.7 compared with 12.3 in the vehicle control) and therefore not attributable to treatment with the test substance. This is supported by the overall value for post-implantation loss, which was not increased, as well as by the number of fetuses per dam, which was even higher in the 150 mg/kg-bw/day dose group than in the vehicle control (11.8 compared with 10.8 in the vehicle control).
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No test substance-related effects on fetal weights were noted. Fetal weights were marginally higher in all groups treated with the test substance than in the vehicle control (4.8 g compared with 4.6 g in the vehicle control, combined data for male and female fetuses, calculated on a litter basis). Since this increase did not show any dose dependency over the wide range of doses applied, it was considered to be incidental.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test-substance effects on the sex ratio of fetuses were noted in any group.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No abnormalities were noted during external examination of fetuses treated with the test substance. In the vehicle control, one fetus with malformation of the head (including upper and lower jaw) and bilateral anophthalmia was noted.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A. Abnormalities and variants: No abnormalities, that were considered to be test substance related, were noted during examination of fetal skeletals and cartilages.
There were 8 fetuses with abnormal skeletal findings as follows: One in the control, two in the 10 mg/kg-bw/day dose group, two in the 30 mg/kg-bw/day dose group and three in the 150 mg/kg-bw/day dose group. The findings noted were confined to common sternebral or vertebral abnormalities (offset sternebrae or bipartite sternebra, dumbbell shaped thoracic vertebral body, unilateral fused zygomatic arch). The low frequencies in this study were considered to be incidental.

B. Stage of development:Occasional statistically significant differences that did occur between groups treated with the test substance and vehicle controls showed no dosage dependency and were caused by higher as well as lower stages of development and therefore considered not to be test substance-related.

C. Cartilage examination of fetuses (abnormal findings and variants): During cartilage examination only 1 fetus with common abnormal cartilage findings was noted (cartelaginous sternebrae 2-5 offset) in the 150 mg/kg-bw/day dose. No abnormal findings or variants were noted in the fetuses of either the control or the 10 or 30 mg/kg-bw/day dose groups.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance related abnormalities were noted during visceral examination of foetuses. During visceral examination of fixed fetuses findings were noted in:
67 of 121 examined fetuses (20 of 21 litters) of the vehicle control
61 of 137 examined fetuses (22 of 22 litters) of the 10 mg/kg-bw/day dose group
63 of 128 examined fetuses (21 of 21 litters) of the 30 mg/kg-bw/day dose group
52 of 118 examined fetuses (19 of 19 litters) of the 150 mg/kg-bw/day dose group
The noted findings (mainly thymus cranially elongated, liver additional lobe(s) of cleft, renal pelvis dilated, testis displaced and umbilical artery left-sided) are common findings for fetuses of this rat strain. Neither the types nor the frequencies of these findings gave any indication for test substance related effects.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Stability, homogeneity and concentrations of the solutions of 2-amino-hydroxyethylaminoanisole sulfate in the vehicle were analytically confirmed.The mean concentrations of the test samples analysed were 102.0 and 99.7 %, 105.4 and 97.9 % as well as 100.3 and 102.2 % of the nominal values for the three dose groups analysed twice at the start and end of the dosing period, respectively.

Applicant's summary and conclusion

Conclusions:
Administration of 2-amino-4-hydroxyethylamino anisole sulfate to female HanBrl:WIST (SPF) rats by oral gavage from Day 6 to Day 20 post coitum at dose levels of 0, 10, 30 and 150 mg/kg bw resulted in a NOAEL of 30 mg/kg bw/day for maternal toxicity (significant reduction in body weight gain and mean food consumption at 150 mg/kg bw/day).
The NOAEL for developmental toxicity was considered to be 150 mg/kg bw/day, the highest dose administered.
Executive summary:

The developmental toxicity study of 2-amino-4-hydroxyethylamino anisole sulfate was conducted following methods comparable to the OECD Guideline 414 (Prenatal Developmental Toxicity Study).

Female HanBrl:WIST (SPF) rats were used in this study. Male and female rats were placed together for mating during the cohabitation period. Day 0 post coitum was designated by the presence of spermatozoa in a vaginal smear or a copulation plug . Mated females were assigned randomly to the following treatment groups of 22 females each:

Group 1: Vehicle control (bi-distilled water)

Group2: 10 mg/kg bw/day (low dose)

Group 3: 30 mg/kg bw/day (mid dose)

Group 4: 150 mg/kg bw/day (high dose)

The test substance was administered to mated female rats by oral gavage once daily from Days 6 through 20 post-coitum. Control animals were treated with bi-distilled water alone. The female rats were observed daily for clinical signs, abortions and deaths. Body weights were recorded from Day 0 to Day 21 post coitum. Food consumption values were recorded at 3 day-intervals on Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum. All rats were sacrificed by CO2 asphyxiation on Day 21, and a macroscopic examination was performed. The number of corpora lutea in each ovary was recorded. The uterus of each rat was weighed and examined for pregnancy, number of implantations, live and dead foetuses and early and late resorptions.

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

In this rat teratogenicity study reduced food consumption and body weight gain were noted in dams of the high dose group (150 mg/kg bw/day) during GD 6 to 20, indicating maternal systemic toxicity of the test item. No treatment-related effects were noted in foetuses up to the highest test dose of 150 mg/kg bw/day. Thus, a NOAEL of 30 mg/kg bw/day for maternal effects and a NOAEL of 150 mg/kg bw/day for embryo-foetal effects were deduced for

2-amino-hydroxyethylamino-anisole sulfate.

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