Sodium 1H-benzotriazolide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1H-1,2,3-Benzotriazole

IUPAC Name:

1H-1,2,3-Benzotriazole

Constituent 2

Reference substance name:

Benzotriazole

EC Number:

202-394-1

EC Name:

Benzotriazole

Cas Number:

95-14-7

Molecular formula:

C6H5N3

IUPAC Name:

1H-Benzotriazole

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Sponsor, Batch No. 111254/112649
- Purity, including information on contaminants, isomers, etc.: 99.87%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: unknown
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: no information stated
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: no information stated
- Reactivity of the test material with the incubation material used (e.g. plastic ware): no information stated

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats are commonly used and recommended to assess toxicity. A large number of
publications and sufficient historic data of the test facility are available.

Sex:

male/female

Details on test animals or test system 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

Details on mating procedure:

- M/F ratio per cage: 1.0
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: single

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Depending on the female performance at least 46 days:

Frequency of treatment:

daily

Details on study schedule:

14 days pre-mating
up to 14 days until mating
an average of 21 days of gestation
between 8-14 days of lactation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

12.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

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

Parental animals: Observations and 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

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, no maximum set for pups/litter.

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

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals approximately four weeks post-mating
- Maternal animals: All surviving animals between days 8 and 14 post-partum

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively:
Epididymides, Testes, Ovaries, Uterus, Adrenal glands, Lymph nodes, Heart

Postmortem examinations (offspring):

no postmortem examinations of offspring

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.

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%).

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.

Reproductive indices:

not calculated

Offspring viability indices:

not calculated

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

From day 7 onwards, either test item treated animals as well as the animals of the vehicle
control group had soft faeces. This was observed throughout the whole in-life phase and was
most likely a side effect of the choice of PEG as vehicle.
From day 5 onwards, individual animals treated with the high and the medium dose of the
test item started wiping their mouth through the cage bedding immediately after application.
This was observed with increasing incidence throughout the treatment period and in nearly
all animals of this dose groups towards the end of the in-life phase.
After 15 days of treatment individual animals of the high dose group started salivating heavily
after application. This was also observed with lower markedness for individual animals
treated with the medium dose of the test item.
Occasional bleeding of mucous membranes at nose and mouth was observed in individual
animals of the high and the medium dose groups. In a few instances respiratory sounds were
observed in individual animals of all test item treated groups after application.
Due to the augmented incidence of all symptoms, especially in the animals treated with the
high dose of the test item, it could be estimated with high probability that the test item causes
discomfort after application of the higher concentrations. Hence, a test item related effect
could not be excluded. No further abnormalities concerning general clinical signs or
behaviour were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

On day 1 one female animal of the high dose group (E660) died immediately after
application. Necropsy of the animal showed that an application error was casual with high
probability.
On day 6 one male animal of the vehicle control group (E656/0) was found dead. The
carcass was found more than 8 hours after death and condition of the carcass did not allow
for necropsy. However, a test item related effect was most unlikely.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Both, the mean body weight as well as the mean body weight gain was comparable between
all female animals treated with the test item and the vehicle control. All values were within
normal range for female rats of this strain and age. Occasional differences especially
towards the end of the gestation phase were assumed to be of natural origin based on the
pregnancy status of the animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Although moderately varying, no test item related tendency could be observed for the food
consumption of the test item treated female animals when compared to he animals of the
vehicle control group. Difference between days 20 and 4 pp were most likely caused
by the physiological conditions of the animals during gestation and lactation period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, non-treatment-related

Description (incidence and severity):

An increase of water consumption (between 9,3 % and 22,0 %) could be observed for the
female animals treated with the high dose of the test item from day 8 onwards when
compared to the vehicle control animals. A similar tendency could be observed for
the animals treated with low dose of the test item from the beginning of the gestation phase
onwards (between 6,0 % and 20,5 %). Conversely the animals treated with the medium dose
of the test item had slightly reduced water consumption throughout the whole gestation
phase (between 5,2 % and 12,5 %).
The water consumption of all male test animals was within normal range for male rats of this
strain and age. No test item related tendency could be observed.
Summarised, no test item related tendencies regarding water consumption of all test item
treated animals (male and female) could be observed throughout the whole post-mating
phase when compared to their respective vehicle control animals. All fluctuations observed
were most likely of naturally origin.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The ovaries, testes, and epididymis from a total of 46 male and female rats (high dose: 12
male/ 11 female; vehicle control: 11 male/ 12 female, see chapter 4.1.2) and all other organs
showing macroscopic lesions of all adult animals were examined histopathologically.
All microscopic findings recorded in the reproductive tract (ovaries, uterus, testes and
epididymides, respectively) of both examined groups (control, high dose) were considered to
be due to pathophysiologic post-partal alterations (females), or were regarded to be
spontaneous in nature and within the normal background pathology commonly seen in rats of
this age. Differences noted between the control and the high dose groups were regarded as
random events. In particular, no specific alteration of the sperm morphology was noted in the
treated males, when compared with the control males.
Coincidental findings in a small number of control and/or test item treated animals were:
Testes: One animal of the high dose group and one animal of the vehicle
control group had tubular atrophy in the testes. One animal of the
vehicle control group had multinucleated giant cells in the testes.
Epididymides: Seven out of twelve animals of the high dose and seven out of
eleven animals of the vehicle control group had mononuclear cell
infiltrations in the epididymides and one animal of the vehicle
control group had aspermia.
Ovaries: One animal of the vehicle control group had a haemorrhage focal
in its ovaries. No further abnormalities were detected within all
animals treated with the high dose of the test item or the vehicle
control group.
Uterus: Eight out of twelve animals of the vehicle control and all eleven
animals of the high dose group had fibrosis in the uterus.
Other organs: Most of the lymph nodes investigated histopathologically showed
histiocytosis, plasmocytosis, erythrophagocytosis and germinal
center reactions, typical indications for local inflammatory
reactions. This was observed in animals of all dose groups and
thus associated less with irritating effects of the test item than
rather with the procedure of application itself.
One male animal of the high dose group had an atrial coagulum.
The adrenals of individual animals showed congestions.
The thymi of individual animals showed hemorrhages or tubular
structures.



The type, incidence and severity of all microscopic findings observed did not indicate a
relationship to the treatment with the test item. These alterations were regarded to be
spontaneous in nature and within the normal background pathology commonly seen in rats of
this strain and age.
Under the conditions of this study, the daily oral administration of the test item “1,2,3-
Benzotriazole-REACH 01” at doses of 12,5, 50 or 200 mg per kg body weight for a treatment
period of at least 39 days did not produce any evidence of pathomorphological findings that
were considered to be due to a toxic effect of the test item.

Histopathological findings: 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

Description (incidence and severity):

During first and second mating phase, no apparent differences occurred regarding the
evidence of copulation and the number of females that achieved pregnancy (Tab. 5). Most
animals conceived within the first five days. One animals of the high dose group and three
animals of the vehicle control group conceived between days 6 and 14 of mating period.
Pregnancy lasted 21 days (n= 2), 22 days (n= 30) or 23 days (n= 13).
Eight animals (E663; E668 and E669 [high dose]; E678 [medium dose]: E687 and E689 [low
dose]; E696 and E699 [vehicle control]) only became pregnant after second mating.
Three female animals of the high dose group delivered one (E664; E667) or two (E665)
stillbirths. One female of the low dose group (E682) gave birth to one stillbirth. None of the
stillborn was deformed or had developmental delays. Although not statistically significant all
stillbirth were within test item treated animals (four at the high dose and one at the low dose
group). Nonetheless, a generally low number of stillbirth was observed during this study. Two
animals of the high dose group (E658 and E665) and two animals of the low dose group
(E687 and E688) had post-natal losses on day 1pp (pups were not found on day 1pp). Two
animals of the vehicle control group had either one (E697) or two (695) dead pups in their
cages on day 4pp.
Two animals gave birth to their litters on day 13 (E663 [high dose]) or day 15 (E689 [low
dose]) of their gestation phase although no sperm plug was detected during mating phase.
All pups were of normal size and appearance, thus it could be assumed that sperm plugs
were missed.
One animal of the vehicle control group (E705) showed signs for delivery on day 22 of its
gestation phase. However, no pups could be found. The animal went into necropsy on day 2
pp. Two implantation sites were counted in the left uterus horn. The mammary glands of the
animals were not developed.
No differences between the dose groups were detectable regarding the number of dams with
both, live young born at day zero (d0, day of birth) and alive pups at day four (d4) of
lactation. The mean numbers per dam of corpora lutea, implantations and live pups at d0 and
d4 were normal for Wistar rats, and no statistically significant differences between the dose
groups were found. In general, the mean pup weight of the rats born in the high dose group
was lower when compared to the vehicle control group. Therefore, data were analysed
additionally by Dunnett’s post-hoc t-test after One-Way ANOVA. The analysis returned no
statistical significance. Four stillbirths were ascertained in the high dose group, one was
found in the low dose group. Nonetheless, the amount of stillbirth was low in general. A test
item related effect could thus be excluded with high probability.
No further abnormalities were detected during gestation and lactation phase.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

> 200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

no detailled description available/necessary

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

no detailled description available/necessary,
in all dose groups and control, a few pups (between 0 and 6) died in the observed 4 days.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

no detailled description available/necessary,

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No abnormal pups were observed in any dose group or in the vehicle group

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Observations	Values
	High dose	Medium dose	Low dose	Vehicle
	200 mg/kg BW	50 mg/kg BW	12.5 mg/kg BW	-
Pairs started (N)	11	12	12	12
1^st mating
Females showing evidence of copulation (N)	9	12	11	12
Females achieving pregnancy (N)	8	10	10	10
Conceiving days 1 – 5 (^5) (N)	7	10	10	7
Conceiving days 6 and more (^1) (^5) (N)	1	0	0	3
2^nd mating
Females showing evidence of copulation (N)	2	1	1	2
Females achieving pregnancy	3	1	2	2
Conceiving days 1 – 5 (^5) (N)	2	1	1	2
Conceiving days 6 and more (^1) (^5) (N)	0	0	0	0
Totals 1^st and 2^nd mating
Females achieving pregnancy (N)	11	11	12	12
Conceiving days 1 – 5 (^5) (N)	9	11	11	9
Conceiving days 6 and more (^1) (^5) (N)	1^2	0	0^2	3
Pregnancy ≤ 21 days (N)	0	0	0	1
Pregnancy = 22 days (N)	9	7	6	8
Pregnancy = 23 days (N)	1	4	5	3
Dams with live young born (N)	11	11	12	11
Dams with live young at day 4pp (N)	11	11	12	11
Implants/dam (mean)	12,7	11,8	11,3	11,2
Live pups/dam at birth (mean)	11,7	12,0	10,8	11,3
Live pups/dam at day 4 (mean)	11,5	12,0	10,6	11,0
Litter weight at birth (mean)	66,8	73,6	67,0	69,5
Litter weight at day 4 (mean)	111,3	121,3	109,2	111,3
Pup weight at birth (mean)	5,7	6,2	6,3	6,2
Pup weight at day 4 (mean)	9,7	10,1	10,5	10,2
No. of pups
Live pups born day 0 (count)	129	132	129	124
Stillborn (count)	4	0	1	0
Total of pups born day 0 (count)	133	132	130	124
Stillborn (%)	3,01	0,00	0,77	0,00
Pups alive day 4	127	132	127	121
Sex ratio
Sex Ration day 0 (total numbers M/F)	59/70	74/58	52/77	52/72
Sex ratio day 0 (mean)	0,84	1,28	0,68	0,72
Sex ration day 4 (total numbers M/F)	68/59^3,4	75/57^3	58/69^3,4	52/69^3,4
Sex ratio day 4 (mean)	1,15	1,32	0,84	0,75

¹ last day of mating period, ² individual animals delivered although no sperm plug was detected 

³ differences to previous rate due to errors at sexing, ⁴ differences due to post-natal losses 

⁵ differences in sum may occur in case an animal achieved pregnancy without sperm plug detected

Observations	Values
					Dosage (units)	High dose	Mid dose	Low dose	Vehicle
200 mg/kg  BW	50 mg/kg  BW	12,5 mg/kg  BW	-
				ABNORMAL PUPS
Dams with 0	11	11	12	12
Dams with 1	0	0	0	0
Dams with ≥ 2	0	0	0	0
LOSS OF OFFSPRING
Pre-implantation (corpora lutea minus implantations)
Dams with pre-implantation loss (count)	11	11	11	10
Pre-implantation loss (mean/group)	3,1	2,9	2,9	3,0
Females with 0	0	1	1	2
Females with 1	2	2	1	2
Females with 2	3	3	4	2
Females with ≥3	6	6	6	6
Pre-natal/post-implantations (implantations minus live birth)
Dams with pre-natal loss (count)	8	6	4	4
Pre-natal loss (mean/group)	1,2	0,9	0,6	1,1
Females with 0	2	5	8	5
Females with 1	5	3	2	1
Females with 2	2	2	1	1
Females with ≥3	1	1	1	2
Post-natal (live births minus alive at post natal day 4)
Dams with post-natal loss (count)	2	0	2	2
Post natal loss (mean pups/group)	0,2	0,0	0,2	0,3
Females with 0	9	11	10	9
Females with 1	2	0	2	1
Females with 2	0	0	0	1
Females with ≥3	0	0	0	0

 

Applicant's summary and conclusion

Conclusions:

The Reproduction Toxicity was examined in a Screening study.
Up to the highest dose of 200 mg/kg bw/day, no effects on the reproduction 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.