Registration Dossier

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 Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate
EC Number:
403-080-9
EC Name:
Sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate
Cas Number:
92484-48-5
Molecular formula:
C16 H17 N3 O4 S .Na
IUPAC Name:
sodium 3-(2H-1,2,3-benzotriazol-2-yl)-5-(butan-2-yl)-4-hydroxybenzene-1-sulfonate
Details on test material:
- Name of test material (as cited in study report): Tinogard HS
- Physical state: powder
- Analytical purity: 99.4 %
- Lot/batch No.: 0004451239
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: (P) 11 wks
- Weight at study initiation: (P) Males: mean ca. 310 g; Females: mean ca. 201 g
- Housing: 5/cage; 1 male + 1 feamle during mating; individually housed after mating (females)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C
- Humidity (%): 28 - 74%)
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Untill 08 April 2011 formulations (w/w) were prepared daily within 2 hours
prior to dosing. Based on stability information from a similar vehicle (4%
CMC), it was known that formulations were stable for at least 2 hours at
room temperature, and as such, the formulations for this study in 1%
CMC were dosed within 2 hours of being prepared until stability over a
longer period of time could be confirmed by analysis. Formulations were
found to be stable for at least 6 hours in 1% CMC on 08 April 2011. As
such, formulations from 09 April onwards were prepared daily within 6
hours prior to dosing. Formulations were homogenized to a visually
acceptable level. No adjustment was made for the specific gravity/density
of the vehicle, or formulation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the vehicle was chosen based on a previous 28-day study

5 mL/kg body weight/day (bw/d). Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until mating occured
- Proof of pregnancy: [vaginal plug and/or sperm in vaginal smear] referred to as [day 0 ] of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
to a validated method (NOTOX Project 496400, BASF Project 05Y0554/10X255). Samples of
formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of
preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also
determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were
90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was
≤ 10%. Formulations were considered stable if the relative difference before and after storage was
maximally 10%.
Duration of treatment / exposure:
males: 29 days; feamles: 43-55 days
Frequency of treatment:
daily
Details on study schedule:
N/A
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 200 and 800 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the dose levels of a 28-days study
Positive control:
N/A

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes: Mortality / Viability
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
Daily, detailed clinical observations were made in all animals, at least immediately after dosing (on the peak period of anticipated effects after
dosing). The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was
predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs,
only its presence (grade 1) or absence (grade 0) was scored.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Weekly, except for males and females which were housed together for mating. Food consumption of mated females was measured on Days 0,
4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was measured daily from 15 April onwards for males and females due to a suspected treatment related effect. For females, water consumption was conducted daily until Day 20 post coitum and from Days 1-4 of lactation.

OTHER: General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Oestrous cyclicity (parental animals):
N/A
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
[testis weight, epididymis weight, other: Of the males of the control and high dose group, and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes was processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin]
Litter observations:
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:
[yes, for external abnormalities; possible cause of death was determined for pups born or found dead.]
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [Following completion of the mating period (a minimum of 28 days of dose administration)]
- Maternal animals: All surviving animals [females that delivered: Lactation Days 5-7; females which failed to deliver: Post-coitum Day 27 (female with evidence of mating)]

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

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
The numbers of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY / ORGAN WEIGHTS
Cervix
Clitoral gland
Prostate gland
Coagulation gland
Seminal vesicles
Epididymides
Testes
Ovaries
Uterus
Preputial gland
Vagina
All gross lesions
Postmortem examinations (offspring):
N/A
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; manyto-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 3; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 4) was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
Mating index (%); Fertility index (%); Conception index (%); Gestation index (%); Duration of gestation;
Offspring viability indices:
Percentage live males at First Litter Check; Percentage live females at First Litter Check; Percentage of postnatal loss Days 0-4 of lactation; Viability index

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality occurred during the study period. At 800 mg/kg bw/day, salivation was noted for all animals over most days during the treatment period. Salivation was also noted for two males at 200 mg/kg bw/d, but at the limited incidence observed, it was not considered to be toxicologically relevant. Incidental findings that were noted included chromodacryorrhoea, alopecia on the abdominal region and scabs on the neck. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be toxicologically relevant.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No toxicologically relevant changes in body weights and body weight gain were noted.
At 800 mg/kg bw/d males had significantly lower body weight gains on Day 8 of the pre-mating period, and over Days 8-15 of the mating period. Males also had lower absolute body weights on Day 15 of the mating period (corresponding to the end of their treatment period). Conversely, females at 800 mg/kg bw/day had significantly higher absolute body weights from Days 4-17 of the post-coitum period and from lactation Days 1-4. These females also had significantly higher body weight gains on Day 8 of the pre-mating period.
Females at 50 mg/kg bw/d had significantly higher absolute body weights from Days 11-14 of the post coitum period with no corresponding increase in body weight gain percentages. These significant differences were not considered to be toxicologically relevant since the effects were
transient (for body weight gains of either sex at various points during the treatment period) and/or the differences from control values were only slight. In the case of higher weights seen for the females, a body weight loss would have been more likely seen if a treatment related effect were evident. In further support of this, there were no corresponding increases in body weight gains for these females at any point during the post-coitum or lactation periods, despite the absolute body weights being significantly higher than controls.

No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.
Absolute food consumption was higher for females at 800 mg/kg bw/d on Days 0-4 of the post coitum period. This was not considered to be toxicologically relevant, however, as the difference from control values was only slight.

WATER CONSUMPTION (PARENTA ANIMALS)
At 800 mg/kg bw/d, water consumption was higher for males and females during the entire measurement period (not always statistically significant).
The increased water consumption may have been a behavioral adaption in response to the salivation seen for all animals. This finding is considered treatment related, but in the absence of any other toxicologically relevant findings, and in the absence of relevant histopathology findings noted for the previous 28-Day study, the toxicological significance of this finding can be doubted.
Several water consumption values for animal no. 48 (control) and two values for animal no. 64 (200 mg/kg bw/d) were excluded from the calculations because of unrealistically high values. While a cause for these high values could not be determined, they were likely due to leakage of the bottles and do not represent actual water consumption.

REPRODUCTIVE DATA
There was a single non pregnant female at 50 mg/kg bw/d. All other females were pregnant.
No toxicologically relevant effects on reproductive parameters were noted up to 800 mg/kg bw/d.
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Terminal body weights and testes and epididymides weights were similar between control and treated animals at all dose levels.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Incidental findings noted for control and/or treated animals included pelvic dilation of the right kidney, a yellowish, soft nodule on the tail of the right epididymis, tan foci on the right clitoral gland and alopecia on the abdominal region. These findings remained within the background range of findings that are seen among rats of this age and strain, and are not related to treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related microscopic findings.
The spermatogenic staging profiles were normal for all Group 1 (control) and Group 4 (800 mg/kg bw/d) males and for Group 2 (50 mg/kg bw/d) male 19.
No cause for the infertility of Group 2 couple male 19 and female 59 could be established from the sections examined.
The microscopic findings recorded were within the normal range of background pathology encountered in Wistar rats of this age and strain.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
for reproduction and development
Effect level:
800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on salivation and increased water consumption noted for animals at 800 mg/kg

Results: F1 generation

Details on results (F1)

No toxicologically relevant effects on the gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopic findings) were observed.
Gestation:
The gestation index and duration of gestation were unaffected by treatment. The gestation index was 100% for all groups.
Parturition/maternal care:
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed.
Early postnatal pup development:
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.
Mortality:
Three pups of the control group, two pups at 80 mg/kg bw/d, and five pups at 800 mg/kg bw/d were found dead or went missing during the first days of lactation. Missing pups were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality
incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Clinical signs:
Incidental clinical symptoms of pups included purple discoloration of the hind feet, blue spot on the abdomen, scabs on the snout and pale appearance. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were not considered
toxicologically relevant.
Body weights:
Body weights of pups were unaffected by treatment up to 800 mg/kg bw/d.
Macroscopy:
Incidental macroscopic findings of pups that were found dead included absence of milk in the stomach. Incidental macroscopic findings among surviving pups included pale appearance and reddish scabs on the hind legs. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were thus not considered to be toxicologically relevant.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion