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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guidance study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 421
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: needles
Details on test material:
- Name of test material (as cited in study report): 1,2,3-Benzotriazole-REACH 01
- Substance type: organic
- Physical state: solid
- Analytical purity: 99.87 %
- Lot/batch No.: 111254/112649
- Expiry date: November 2012
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: P 10-11 wks
- Weight at study initiation: (P) Males: 294- 351 g; Females: 196 - 225 g;
- Fasting period before study: non
- Housing: females single, males in groups of three
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 to 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+-3°C
- Humidity (%): 30-70%
- Air changes (per hr):10
- Photoperiod (hrs dark / hrs light):12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Instructions for test item preparation:
1. Weigh required amount of the test item into an appropriate vial on an analysis scale.
2. Add PEG400 up to the required volume to produce the application suspension as
stated in the laboratory work sheets.
3. Stir until suspension is a consistent emulsion; vortex if necessary.
4. Stir immediately before each dose will be drawn into the application syringe.
According to the Sponsor the test item is stable in PEG400 for seven days at concentrations
of 12,5 g/l and 50 g/l. The highest concentration of the test item suspension was prepared
once every 6 or 7 days and stored in a dark place at room temperature. Daily, a serial
dilution (1 in 4) of the highest concentration was made to produce the further application
solutions. The test item preparation was intended for an application volume of 4 ml per kg
body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
As the test item’s solubility in water and corn oil is poor, polyethylene glycol 400 (PEG400)
will be used as an organic solvent. PEG400 has already been used as vehicle for the
subacute oral toxicity study of the surrogate substance Methylbenzotriazole. PEG400 is a
commonly used pharmaceutical excipient (e.g. suppositories, capsules or tablets) and also
frequently used in toxicological work to improve the solubility of otherwise poorly soluble
compounds. The LD50 of PEG is 51,3 g/kg body weight, and the maxiumum recommended
daily uptake is a quarter of the LD50.
- Concentration in vehicle: 50 g/l
- Amount of vehicle (if gavage): 4 ml/kg
- Lot/batch no. (if required): K43386003
- Purity: Not applicable
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days or until pregnancy occured
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear] referred to as day 0 of pregnancy
Duration of treatment / exposure:
Depending on the female performance at least 46 days:
Frequency of treatment:
daily
Duration of test:
in total: 57-77 days
14 days pre-mating
up to 14 days until mating
an average of 21 days of gestation
between 8-14 days of lactation
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a previously performed dose range finding study4, the test item was administered at three
doses up to 200 mg/kg body weight over a time period of at least six weeks which produced
no acute toxic effects in the test animals. In accordance with the Study Monitor, 200 mg/kg
was determined as high dose for this study followed by two graduated (1 in 4) serial dilutions
thereof (50 mg/kg, 12,5 mg/kg) assigned as medium and low dose.
The high dose was chosen based on following arguments:
In an acute toxicological assessment quoted in the material safety sheet on the test item,
560 mg/kg wasdetermined as the LD50. A subacute oral toxicity study for the surrogate substance Methylbenzotriazole (study report indicates LD50 value at 720 mg/kg) showed signs of toxicity at a dose level of 450 mg/kg. Thus, 200 mg/kg were determined as high dose for the dose range finding study. Two graduated 1:2 v/v) serial dilutions thereof (100 mg/kg, 50 mg/kg) were assigned as medium and low dose for this assay.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: once weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: once weekly
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:
Examination of the born litter

- External examinations: Yes: all per litter
Statistics:
Spread sheet calculations were performed using Microsoft® Excel® 2011 for Mac.
Food- and water consumption of male animals were documented sorted by experimental
groups, whereas the body weight was documented for each animal individually. Body weight,
food- and water consumption, litter size and litter weight were documented for each female
animal individually.
3.4.1 Descriptive statistics
The arithmetic mean, standard deviation and median were calculated for all grouped
numerical data originating from monitoring the body weight, food- and water consumption,
organ weights (gross pathology) and litter size and weight (for details see appendix). Where
appropriate, detailed column statistics were applied (minimum / maximum data, 25%
quantiles, standard error, upper and lower confidence interval 95%).
3.4.2 Deductive statistics
If appropriate, the respective test item groups were compared to the vehicle group by
assessing statistical significance using a two-tailed unpaired Student´s t-test. For all
calculations, the significance level was set to 0,05.
For some analysis parameters that returned statistical significances in the t-test, further
deductive statistics were applied as outlined in the schematic decision tree displayed in the
appendix. Most statistical hypotheses in this study were best characterised as “many to
one”– a vehicle control vs. three treatment groups, respectively. Therefore the adequate
analysis method was a One-Way ANOVA (Analysis of variance), followed by a post hoc
Dunnett´s t-test. In case a sufficient number of values per group were available a Bartlett´s
test for equal variances was applied on the data. For all calculations, the significance level
was set to 0,05. These further deductive statistics then were performed using Graph Pad
Prism for Mac, Version 5.01. Statistical data and analyses were documented in the appendix.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Gross pathological findings:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
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
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The Developmental Toxicity was examined in a Screening study.
Up to the highest dose of 200 mg/kg bw/day, no effects on the Development were observed
Executive summary:

In the present study toxic effects of the test item 1,2,3-Benzotriazole-REACH 01 at a

maximum dose of 200 mg/kg body weight on the development and reproduction of Wistar

rats after oral administration were under examination.

Mild discomfort throughout the whole application period was observed for the animals treated

with the high and the medium dose of the test item (wiping of nose and mouth through the

cage bedding, salivation after application, bleeding of mucous membranes at nose and

mouth, respirators sounds). A biological and particularly toxicological relevance of those

observations could not be excluded completely.

Regarding the body weight and the body weight gain, no significant differences were

observed between all test item treated animals (male and female) and their respective

vehicle control animals. Occasional differences observed for the females were assumed to

be of natural origin based on the pregnancy status of the animals.

The food consumption of the female animals was not effected by the administration of the

test item whereas the amount of water consumption was either enhanced (high and low dose

group) or reduced (medium dose group) when compared to the vehicle control animals. An

impact of the test item administration on the food and water consumption of the male animals

could not be observed.

The most prevalent findings during necropsy were reddened and swollen lymph nodes and

effects on the digestive system. All other findings were observed with low incidences and

without any test item related tendency. They were thus regarded to be spontaneous in

nature.

Histopathological examination of the ovaries as well as of the epididymides/testes did not

produce any evidence of pathomorphological findings that are considered to be due to a toxic

effect of the test item.

Regarding the reproduction and developmental parameters gathered in this study no

statistical significant changes were observed that were definitely treatment-related.

Reproduction success (achievement of pregnancy after indication of copulation, litter size

and survival rate of pups) was comparable between test item treated animals and the vehicle

control group. While the amount of live young born showed no significant differences in the

high dose group, the mean pup weight of the young born in this dose group was (although

not statistically significant) reduced compared to the vehicle control animals. Nonetheless, in

the absence of other findings regarding the developmental parameters a test item related

effect could be excluded with high probability.

A daily oral administration of the test item 1,2,3-Benzotriazole-REACH 01 to male Wistar rats

at dose levels of 12,5 mg, 50 mg and 200 mg/kg body weight over a time period of 39 to 50

days did not produce any pathological evidence for toxic effects on the reproduction

performance of male rats. However, an effects of the spermatogenesis may not have had an

adequate time to become evident (such as reduced sperm counts affecting the fertility) as

chemical exposure does not cover a complete cycle of spermatogenesis in male test

animals.

A daily oral administration of the test item to female Wistar rats at dose levels of 12,5 mg,

50 mg and 200 mg/kg body weight over a time period of 46 to 70 days did not produce any

pathological evidence for toxic effects on the reproduction performance of female rats

regarding the achievement of pregnancy, litter size and survival rate of pups.