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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to OECD test guideline 421

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
minor changes, e.g. in dosing, with any impact on study results
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
dentification: SULFANILIC ACID TECHNICAL GRADE
Formula: H2N-C6H4-SO3H
Molecular mass: 173,19 g/mol-1
Purity: 99,12%
Batch No.: M001/09
Batch production: 01 October 2009
Appearance: Grey powder (micronised)
Storage conditions: Conventional, ambient temperature
Expiry date: 01. Oct. 2011

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Species: Rattus norvegicus
Strain: Wistar-WU
Gender: 48 males, 48 females
Source: Charles River, Sandhofer Weg 7, 97633 Sulzfeld, Germany
Age at acclimatisation: 9-10 weeks
Health status: Specific pathogen free (SPF)
Pregnancy status females: Nulliparous, non-pregnant
Acclimatisation: 7-10 days

Housing conditions
Clean conventional housing: airing with approx. 10 air changes per hour, room climate 22 ± 3°C at 30-70% relative humidity, (aimed
limits - due to meteorological circumstances, upper limit might have been above 70% on short term), artificial lighting 12 h light/12 h dark.

Caging
Groups of up to three animals in open macrolon cages type 2000P, TechniPlast (size slightly larger than GV-SOLAS Type IV).

Bedding:
Lignocel hygienic animal bedding (J. Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg)

Diet
Maintenance diet for rats and mice, No. 1324 TPF (Altromin Spezialfutter GmbH & Co. KG, 32791 Lage), ad lib.
Dams: Breeding diet for rats and mice, No. 1314 TPF

Water
Sterilised community tap water, ad lib.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
suspension in water

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Although the test item’s solubility in water is poor, results from a preceding dose range finding study indicated that the micronised (<100 μm) test item could be applied as a colloid suspension in water at the three dosages specified in this study report.
- Amount of vehicle (if gavage): 4 ml/kg bw
Details on mating procedure:
During the mating phase, the female were placed with the same male until indications formating were detected. Each morning, the females were examined for the presence of sperm or a vaginal plug. Day 0 of pregnancy was defined as the day a vaginal plug or sperm was
found.
Females showing no evidence of copulation were regrouped with proven sires for a second mating phase. Dosing was continued in both sexes
during the mating period.
As mating was not efficient in a first phase in the control and all does groups a second mating phase was included.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
males: at least 6 weeks, but on average about 8 weeks (due to second mating period)
females: at lest 6 weeks, but on average about 8-9 weeks with a maximum of days (due to second mating period)
Females were dosed two weeks prior to mating, covering at least two complete oestrous cycles, the variable time to conception, the duration of pregnancy and at least four days after delivery, up to and including the day before scheduled termination of the in-life phase.
Therefore the duration of the study following acclimatisation depended on the female performance: 14 days pre-mating, up to 14 days until mating, an average of 21 days of gestation, and a minimum of 4 days of lactation.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
62.5 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
In the previously performed dose range finding study and subacute oral toxicity study (OECD 407), the test item was administered in three dosages up to 1000 mg/kg body mass over a time period of 28 or at least 40 days respectively and produced irritating but no observable toxic effects in the test animals. In accordance with the sponsor, 1000 mg/kg was determined as high dose for this study.
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage-side observations to detect signs of illness or reactions to treatment, moribund animals or fatalities were conducted

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekl

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: twice weekly
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight, didymal sperm reserve
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain

GROSS EXAMINATION OF DEAD PUPS:
no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [after at least 6 weeks]
- Maternal animals: All surviving animals [day 4 post-apartum]

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

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examinations were performed on the ovaries, testes and epididymides (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) of the animals of the highest dose group and the control group. The
other preserved organs would have been examined if necessary.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Except one male rat (death prematurely on day 41 of the in life phase due to an application error9), no mortality was observed.
Beginning after the first application of the high dose (1000 mg/kg body mass), animals salivated heavily and wiped their mouth in the cage bedding material for approximately 30 seconds immediately after application, indicating mild discomfort. Some animals from both sexes of the high dose
groups showed spontaneous oronasal bleedings during the first week of application. This effect was occasionally observed throughout the course of the study. In absence of other effects, the bleedings were considered an indication of irritating effects of the test item and of
no toxicological relevance.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

ORGAN WEIGHTS (PARENTAL ANIMALS)

GROSS PATHOLOGY (PARENTAL ANIMALS)

HISTOPATHOLOGY (PARENTAL ANIMALS)

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a GLP-study according to OECD test guideline, both the parental NOAEL and the NOAEL for developmental and reproductive toxicity were 1000 mg/kg bw.
Executive summary:

Aim of the study:

The aim of this GLP-study was to assess data on reproduction and developmental toxicity of the test substance, suspended in water, after oral administration to Wistar rats (according to OECD test guideline 421).

Experimental model:

The test substance was administered daily by oral gavage at dose levels of 62,5 (50), 250 and 1000 mg/kg body mass (BM) / day to 12 male and 12 female Wistar rats each. Another 12 male and 12 female rats received the same volume of water as vehicle control. All liquids were administered to each animal at 4 ml / kg bw. Males were dosed daily for a minimum of fr weeks, including the day before the scheduled termination of the in-life phase. This included a minimum of two weeks of dosing prior to

mating and continued throughout the mating period until the study was terminated. Females were dosed two weeks prior to mating, the variable time to conception, the duration of pregnancy and at least four days after delivery, up to and including the day before scheduled termination of the in-life phase. Females showing no evidence of copulation were re-grouped with proven sires for a second mating phase. Dosing was continued in both sexes during the mating period.

Assessed parameters:

During the in-life phase, all animals were monitored for fatalities, general clinical signs, body mass as well as group food and group water consumption. At the end of the in-life phase, all animals were sacrificed humanely and examined by gross necropsy. Organ mass was recorded according to the study plan, tissues and organs selected therein were preserved and processed histologically. A histopathological examination was conducted on samples from the high dose groups and the vehicle groups.

Results:

After oral administration of the test substance for 41 to 79 days (mean: 59 days due to a second mating period), the monitored animals showed no abnormalities regarding the clinical signs, body mass as well as food and water consumption.

At gross necropsy, no abnormalities were found that could be related to the administration of the test item. The organ mass gave no evidence for toxicological effects of the test item on the sexual organs of Wistar rats. The histomorphological examination of selected rat organs of the male and female genital system (24 male and 24 female) did not reveal morphological lesions related to the test item.

There was no morphological difference between the vehicle control group and the groups subjected to the high dose of the test item. The diagnosed minimal to mild atrophy of the germinative epithelium in the testes of the vehicle control group and the high dose group

were interpreted as coincidental findings. All the histomorphological findings recorded in this study are commonly encountered in rats of this strain and age. Type, incidence, and severity of the lesions recorded were not increased in the treated animals as compared to the control animals. The vast majority of data regarding development and reproduction indicated no difference between the animals treated with the test item and the vehicle control. None of the average data points were overall extremely out of range for rats of this strain and age, and no pattern of significant abnormalities occurred. Very few observations among the assessed parameters were considered as possibly test item induced effects. Noteworthy were the statistically not significant, mild effects on litter mass of the high dose group and on pre-implantation loss of the medium and high dose group. Similarly there was a mild tendency of relatively low total numbers of pups born with increasing test item dosage and a low mean number of live pups per dam at day 4 pp. The combination of these findings suggested a mild influence of the high dose of the test item on the respective reproductive parameters. However, findings of this mild nature are in favour of an adaptive response rather than a direct toxic effect of the test item.

Conclusion:

A daily oral administration of the test substance to Wistar rats at a dose level of 62,5 (50), 250 and 1000 mg/kg body mass over a time period of 41 to 79 days resulted in very few mild systemic and local effects in test animals treated with the high dose (1000 mg/kg) but did not produce any pathological evidence of local or systemic toxicity of the test item or any toxic effects on the development and reproduction.