Sodium 1H-benzotriazolide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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

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

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

Dose / conc.:

12.5 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

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.

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

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: 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.

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

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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

 

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

only one study of an analogue available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The performed screening test for Benzotriazole shows no adverse effects up to 200 mg/kg bw/day, a NOAEL of 200 mg/kg bw/day can be derived. The read across from Benzotriazole to Sodium Benzotriazolate is feasible.

The results of the screening test are of limited use for the characterisation of this hazard as stated in the OECD Guideline:

"This test does not provide complete information on all aspects of reproduction and

development. In particular, it offers only limited means of detecting post-natal manifestations of prenatal

exposure, or effects that may be induced during post-natal exposure."

For the risk characterisation it is possible to use the test results, if the exposure is clearly below the NOAEL.

Short description of key information:
In a Screening Test for toxicity to reproduction and development of the analogue Benzotriazole, performed according to OECD Guideline 421,
Doses of 12.5, 50 and 200 mg/kg bw/day were tested.
No adverse effect on the reproduction or development of the test species (rats) was observed for the analogue.
Sodium Benzotriazolate has similar reproductive and developmental dose toxicity compared to the analogue Benzotriazole resulting in similar no observed adverse effect level (NOAEL), because the source chemical Benzotriazole is sufficiently similar to read-across towards Sodium Benzotriazolate.

Justification for selection of Effect on fertility via oral route:
A well performed study according to OECD Guidelines of an analogue is chosen.

Effects on developmental toxicity

Description of key information

In a Screening Test for toxicity to reproduction and development of the analogue Benzotriazole, performed according to OECD Guideline 421,
Doses of 12.5, 50 and 200 mg/kg bw/day were tested.
No adverse effect on the reproduction or development  of the test species (rats) was observed for the analogue.

In an oral pre-natal study OECD 414, administration of the analogue 1,2,3-Benzotriazole at doses 36, 120 and 330 mg/kg body weight/day from implantation to the day prior to scheduled caesarean section (day 5 to day 19 post-mating inclusive) resulted in maternal and developmental toxicity (i.e. post implantation loss, external and skeletal malformations). Under the conditions of this study, the no-observed-adverse-effect-level (NOAEL) of 1,2,3-Benzotriazole for maternal and developmental toxicity was considered to be 36 mg/kg bw/day and for embryo-fetal developmental toxicity was considered to be 120 mg/kg bw/day.

Sodium Benzotriazolate has similar reproductive and developmental dose toxicity compared to the analogue resulting in similar no observed adverse effect level (NOAEL), because the source chemical is sufficiently similar to read-across towards Sodium Benzotriazolate.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2019 - April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Connect Chemicals GmbH, 19303
- Expiration date of the lot/batch: 02.2021
- Production date: 03.2019



STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
-Stability of the test item in the vehicle prepared at concentrations of 12 and 110 mg/mL was
confirmed following storage for 7 days at room temperature (the temperature range, 20 – 25
°C) during method validation study. In addition, homogeneity analysis was performed for
formulations of 12 and 110 mg/mL after 4 days of storage after re-mixing

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The Lab Animals Breeding Center “Pushchino”, Branch of
Institute of Bioorganic Chemistry RAS:
Nauki 6, Puschino, Moscow region, Russia 142290
- Age at study initiation: Approximately 11-12 weeks old at the initiation of dose
administration on G5 (day 1)
- Weight at study initiation: 233 ± 15 g, N = 93
- Fasting period before study: no
- Housing: After identification and until mating, all females were housed by groups. Males were housed
alone, and females cohabited with a male in the home cage of the male (2:1). After confirmed
mating, dams were housed alone until euthanasia on gestation day 20
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: ca 7-8 weeks, The animals were received at the age of 4 weeks
by separate litters to avoid of sibling mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30-70%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/ 12

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The Sponsor provided the test item as a neat solid substance. Calculations for the preparation of formulations were based on the dose (36, 120 and 330 mg/kg bw) and administered volume (3 mL/kg body weight), were documented in Excel spreadsheets, and maintained in the study file as printouts.
The test item was dissolved in the required volume of the vehicle (Kollisolv® PEG E 400) in
order to achieve the concentrations of 12, 40, and 110 mg/mL and then homogenized using a magnetic stirrer.
Test item formulations were prepared every four days, aliquoted to the required volumes of days of the administration, and stored in tightly closed glass jars at room temperature in the
dark. For the control group, the required volume per day of Kollisolv® PEG E 400 was placed in a labeled jar.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on preliminary studies, the vehicle was chosen
- Concentration in vehicle: 12, 40, and 110 mg/mL
- Amount of vehicle (if gavage): 3 mL/kg body weight
- Lot/batch no. (if required): BCCB1456
- Purity: not stated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the test item in the vehicle prepared at concentrations of 12 and 110 mg/mL was
confirmed following storage for 7 days at room temperature (the temperature range, 20 – 25
°C) during method validation study. In addition, homogeneity analysis was performed for
formulations of 12 and 110 mg/mL after 4 days of storage after re-mixing.
Analysis of formulations for homogeneity and concentration during dosing period was
conducted in the test facility at the beginning, in the middle and at the end of
in-life phase using a validated method.
For homogeneity analysis, quadruplicate samples (approximately 0.1 mL of each) were
collected from the top, middle and bottom strata of each dosing formulation prepared during
the study.
For concentration analysis, quadruplicate samples were collected from the middle stratum of
each dosing formulation (including the vehicle control group) prepared during the study.
Samples collected from the mean stratum for homogeneity analysis used for this purpose.
A pair of quadruplicate samples from each strata was used for analysis, the other pair was
stored as back-up samples at room temperature in tightly closed flasks, analyzed if necessary
based on primary assays to verify concentration and were discarded after the study director's
approval of the analytical results.
Acceptance criteria for the formulations analysis are based on the test item in vehicle
composition as a suspension. For stability, the mean concentration of formulation samples of
12 and 110 mg/mL after 4 days of storage and resuspension should be within the acceptable
range of the target concentration (85-115% with RSD<10%), and within the range 85-115% of
the time zero point. The actual concentration of analyzed samples collected from the mean
stratum of formulations should be within the range of 85% - 115% of the target concentration.
The acceptance criteria for homogeneity are RSD<10% with the mean concentration within
85% to 115% of the target concentration.

Details on mating procedure:

After identification, females were monitored to an estrous cyclicity daily during 3-5 days. Females with
clear stages of estrous cycle in vaginal smear were cohabited with a male (avoiding siblings mating),
2:1 until mating. Positive evidence of mating was confirmed by the presence of a vaginal copulatory
plug or the presence of sperm following a vaginal lavage. Each mating female was examined daily on
the morning. The day when evidence of mating was identified is termed as gestation day 0 (G0).

Duration of treatment / exposure:

15 days, from day 5 to day 19 (including) of post mating (G5-G19)

Frequency of treatment:

daily

Duration of test:

Until Gestation day 20

Dose / conc.:

36 mg/kg bw/day (nominal)

Dose / conc.:

120 mg/kg bw/day (nominal)

Dose / conc.:

330 mg/kg bw/day (nominal)

No. of animals per sex per dose:

ca 25

Control animals:

yes, concurrent vehicle

Details on study design:

Before the beginning of the treatment period, the females with confirmed mating were
allocated to the groups, according to a stratification procedure, so that the average body
weight of each group did not statistically differ. Females with the same day of gestation were
allocated to a different group.

Maternal examinations:

Cage Side and Clinical Observations
All rats were observed twice daily, once in the morning and once in the afternoon at the same time,
for morbidity and mortality. Each female was also observed for signs of toxicity approximately 15-45
minutes following dose administration. In addition, the presence of findings at the time of dose
administration was recorded for individual animals.

Body Weights (F0)
Individual female body weights were recorded during animal identification, at day of confirmed mating
and group assignment (gestation day 0), on the first day of dose administration (gestation day 5), and
at three-day intervals thereafter (gestation days 8, 11, 14, 17, and 20 as the day of euthanasia). Body
weight value on gestation day 20 was corrected for gravid uterine weight to calculate maternal body
weight change.

Food Consumption
Food consumption was assessed for each female quantitatively as g/kg of body weight/day by
weighing of feeder (cage lid) at the beginning of the day and 24 hours after. Food consumption was
recorded prior to the initiation of dose administration (gestation days 0-1), and at three-day intervals
thereafter (gestation days 4-5, 7-8, 10-11, 13-14, 16-17 and 19-20).

Scheduled Euthanasia
On gestation day 20, all females were euthanized by anesthesia (Zoletil® / Xyla®, i.m.)
followed by terminal blood sampling for hormones assay and subjected
to hysterectomy.
The weight of the gravid uterus was recorded for each pregnant female (with at least one live
fetus). Uterus were examined. Ovaries were examined to determined number of corpora lutea.
Each dam was examined macroscopically, thyroid gland was collected in 10% neutral formalin
(in complex with trachea and esophagus) and weighed after fixation.

Necropsy of Females and Examination of Uterine Content
A complete necropsy was conducted on all females at scheduled termination. Necropsy
included examination of the external surface of the body, all orifices, the cranial cavity, the
external surface of the brain, and the thoracic, abdominal and pelvic cavities including viscera.
Hysterectomy and examination of uterine content was done for all females. Ovaries were
examined to determined number of corpora lutea. Gravid uterine weight was collected at
scheduled necropsy. Uteri, which appear non-gravid by macroscopic examination, were
opened and placed in 10 % ammonium sulfide solution for detection of early implantation loss.
Thyroid glands were preserved (in scheduled or euthanized in extremis females) and weighed
after fixation (scheduled euthanized).

Blood Sample Collection for Hormones Assay
Blood samples were collected from all surviving females at the scheduled necropsies
(as part of euthanasia on day 20 of post-mating).
Animals were not fasted prior to blood collection. The blood was collected terminally
following anesthesia (Zoletil® / XylaVET®) from the caudal vena cava after
laparotomy using a syringe with 23G needle. Blood collection was done on the first
part of the day (within 10:00 – 13:00 hours) in randomized order to avoid bias.
The blood sample was placed in a tube without anticoagulant. The blood was allowed
to clot for 50 min and centrifuged (1600 x g, 4 °C, 15 min) for serum separation.
Serum from each animal was divided into 6 aliquots (for two aliquots for each of T4,
T3 and TSH analysis) and immediately frozen at –70 °C until assayed.

T4, T3 and TSH Assay
Thyroid hormones (thyroxin (T4), triiodothyronine (T3), and thyroid stimulating
hormone (TSH)) were assayed in serum from all pregnant females (with at least one
fetus) by competitive inhibition enzyme immunoassay technique using relevant ELISA
kits (see below) and Multiskan™GO Microplate Spectrophotometer (Termo Scientific)
and according to standard procedure of manufacturer and SOP of BTL BIBC RAS.

Microscopic Examination of Thyroid Gland
Thyroid gland of all females euthanized at the scheduled necropsy was trimmed,
embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined
microscopically.

Ovaries and uterine content:

The weight of the gravid uterus was recorded for each pregnant female (with at least one live
fetus). Uterus were examined. Ovaries were examined to determined number of corpora lutea.
Each dam was examined macroscopically, thyroid gland was collected in 10% neutral formalin
(in complex with trachea and esophagus) and weighed after fixation.

Fetal examinations:

After cesarean section, all fetuses were subjected to external examination. Half of the fetuses
from each litter was examined for skeletal abnormalities and the remaining for soft tissue
alterations.
The fetal findings were described according to the harmonized terminology of the International
Federation of Teratology Societies (IFTS) [1, 2] without categorization and classified as
malformations or variations:
 malformation (major abnormality) refers to structural change considered detrimental to the
animal (may also be lethal) and is usually rare;
 variation (minor abnormality) refers to structural change considered to have little or no
detrimental effect on the animal; may be transient and may occur relatively frequently in
the control population.
The reproductive tract was examined with particular attention, and external sex was compared
with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue
malformations). In addition, male fetuses were examined for undescended testes.

External Examination, Body Weight and AGD
Each live fetus was weighed and sexed with measurement of anogenital distance
(AGD). All fetuses were subjected to a detailed external examination for gross
anomalies with a recording of malformations and variations or non-classified findings.
Fetuses were then anaesthetized by subcutaneous injection of Zoletil® + XylaVET®
mixture and fixed in 96 % ethanol (approximately one-half of litter) for skeleton
examination or Bouin’s fixative (remainder of litter) for soft tissues examination.

Skeletal Examination
Approximately half of the live fetuses in each litter was fixed in ethyl alcohol,
eviscerated and skinned for subsequent double staining of cartilage and bone with
alcian blue and alizarin red.
A detailed examination of the skeleton was done and included the observation of all
the bone and cartilage structure of the head, spine, rib cage, pelvis, and limbs.
During evisceration, the reproductive tract was examined and external sex was
compared with internal (gonadal) sex; an indication of incomplete testicular descent
was noted in male fetuses.

Soft Tissues Examination
The remaining live fetuses in each litter were fixed with Bouin’s fluid. A detailed soft
tissue examination was performed according to a free-hand serial sectioning
technique, which will include the observation of all the organs and structures of the
head, neck, thorax and abdomen.
Organs within the abdomen were examined in unsectioned abdominal region with
particular attention on reproductive tract; an indication of incomplete testicular
descent was noted in male fetuses.

Statistics:

All statistical tests were performed using Microsoft Excel (descriptive statistics) and statistical software
Statistica for Window v.7.1 to compare the treated groups to the control group. Descriptive statistics (mean,
standard deviation (S.D.), and N) are presented for all measurement data and shown in the summary
tables. The litter is accepted as an experimental unit for statistical analysis.
Continuous data variables (mean body weights and food consumption data) were analyzed by multi-factor
analysis of variance ANOVA-2, followed by the Duncan test, to determine inter-group differences. Former
implantation sites, number of corpora lutea, implantation loss indices, hormones concentration value,
uterine, and thyroid weights were analyzed by parametric one-way analysis of variance (ANOVA). If the
results of the ANOVA were significant (p<0.05), Dunnett's test is applied to the data to compare the treated
groups to the control group. The t-test was used additionally to compare each dose group with the control
value. Offspring sex ratio data, AGD value, and mean value of affected fetuses per litter were subjected to
the Kruskal-Wallis nonparametric ANOVA test to determine intergroup difference. If the results of the
ANOVA were significant (<0.05), Dunn's test is applied to the data to compare the treated groups to the
control group.
The fetal body weight was analyzed by sex as well as for both sexes combined using a one-way analysis of
variance (ANOVA) as described above. Additionally, statistical analysis for fetal body weight was done
using analysis of covariant with litter size as a covariant.
Descriptive data, percentage values, and pathomorphological data were analyzed by Fisher's Exact Test
and additionally by Chi-square test (when a tendency to a difference was observed).

Indices:

Parameters Calculation / Note
Pregnant females alive at termination (N)
Body weight at necropsy (g)
Gravid uterine weight (abs, % of pregnant
female body weight)
Thyroid weight (abs, % of body weight without gravid uterine weight)
Females with total resorptions (N)
Females with all dead fetuses (N)
Females with live fetuses (N)
Corpora lutea (N per animal)
Implantation sites (N per animal)
Pre-implantation loss (N per animal, % of implantation sites) (Number of corpora lutea – Number of implantation sites) / Number of corpora lutea
Fetuses (N per animal)
Live fetuses (N per animal, % of implantation sites)
Dead fetuses (N per animal, % of implantation sites)
Resorptions + Scars (N per animal, % of implantation sites)
Implantation scars (N per animal, % of implantation sites)
Resorptions – early (N per animal, % of implantation sites)
Resorptions – late (N per animal, % of implantation sites)
Post-implantation loss (N per animal, % of implantation sites) (Number of implantation sites – Number of live fetuses) / Number of implantation sites

Historical control data:

none reported

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The test item related clinical findings were observed in the 330 mg/kg bw/day dose group as follows: hypokinesia (12 females), staggering gait (11 females) or flatness as a more severe behavior disorder (6 females), excessive vocalization (2 females), chromodacryorrhea (2 females), ptyalism (2 females), nasal discharge (2 females); eyelid closure, hyphosis, and diarrhea was noted for single females.
These observations were episodic with the recovery of animals within approximately 1.5 – 3 hours after dosing. The higher frequency of clinical signs was noted after first dosing (gestation day 5), as well as at the end of the administration period (gestation days 14, 15, 17, and 19). Over the entire period of the test item administration, the total number of females with clinical findings in the 330 mg/kg bw/day dose group was 14 out of 26 examined (p < 0.001, Fisher Exact test).
In the 120 mg/kg bw/day dose group, one female (No. 18) had focal alopecia on the forelimbs observed beginning from day 9 of dosing, which supposed to be not test item related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There was no morbidity and mortality of females caused by the test item administration.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight of pregnant females administered with the dose of 330 mg/kg bw/day was slightly decreased compared to the control vehicle group at the gestation day 20 (by 5.0%, p < 0.05). The body weight gain in 330 mg/kg bw/day dose group was statistically reduced (p < 0.01) the entire period of administration compared with the control group as well as lower doses.
The reduced body weight gain of pregnant females in the 330 mg/kg bw/day dose group was associated with a smaller weight of the pregnant uterus (by 16 % compared to the control group, p < 0.05). The final maternal body weight without a gravid uterus did not statistically differ from the body weight of control females.
See Table S5 and S6 in 'Any other information on results incl tables' for details.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption in the 36, 120 and 330 mg/kg bw/day group was similar to that in the vehicle control group during all study days.

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

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid Hormones Assay Data
Benzotriazole at the doses of 36, 120, and 330 mg/kg bw/day did not significantly affect the level of thyroid hormones (Thyroxine, Triiodothyronine, and Thyroid Stimulating Hormone) in the serum of pregnant females.
See Table S12 and S13 in 'Any other information on results incl tables' for more details.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The absolute and relative thyroid weight of pregnant females was not significantly changed in all dose test item treated group.
See Tables S6 and S11 in 'Any other information on results incl tables' for details.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All treated females were sacrificed during a scheduled necropsy on post-mating day 20.
During necropsy, there were no gross findings related to the test item administration. One female (No. 94) from the 330 mg/kg bw/day dose group had the uterus hydrometra, which is supposed to be not test item related.
Females No.38 (group 1), No.16, No.42, and No.56 (group 2), No.8, No.33, and No.94 (group 4) were without fetuses in the uterus. In female No.16, two early resorptions were revealed; there were no implantation sites, and scars were observed in non-pregnant females after staining of their’s uterus by ammonium sulfide.
See Tables S9 and S10 in 'Any other information on results incl tables' for details.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

C-cell hyperplasia considered as test item related finding was observed in 3 of 24 females from 120 mg/kg bw/day dose group, and in 4 of 23 females from 330 mg/kg bw/day groups. C-cell hyperplasia was of minimum grade; however, it was not found in the control group, and the increase in occurrence in the high dose group was significant (p < 0.05, Fisher Exact test)
compared with the control group. Although there is no data on the serum level of calcitonin and calcium in pregnant females treated with benzotriazole, C-cell hyperplasia may be considered potentially adverse.
Remaining histological findings (congenital cysts, ectopic lymphoid tissue, and one finding of follicular cell vacuolization) were considered to be incidental not treatment-related findings.
See Table S12 in 'Any other information on results incl tables' for details.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

none reported - see Table S10 in 'Any other information on results incl tables' for more details.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The mean number of corpora lutea and pre-implantation loss did not statistically differ in the test item treated groups compared to the control vehicle group. The mean number of implantation sites was approximately the same among groups.

In the 120 and 330 mg/kg bw/day dose groups, the relative post-implantation loss was
increased compared to the control vehicle group (p < 0.01 and p < 0.05, respectively). The increase of post-implantation loss in these groups was associated with the increase in the absolute and percentage values of early resorptions. Also, the total number of females with post-implantation loss was increased (non-significantly) in the 120 and 330 mg/kg bw/day dose groups. The mean number of fetuses per female was slightly decreased in the 330 mg/kg bw/day dose group (by 6.2 % compared to the control group), but this change was not significant.

See Tables S9 and S10 in 'Any other information on results incl tables' for details.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

no total litter loss was reported - see Table S10 in 'Any other information on results incl tables'

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

a significant difference in early resorptions is reported for the 120 and 330 mg/ kg bw/day dose group (p < 0.01 resp. p< 0.05) - - see Table S10 in 'Any other information on results incl tables'

Dead fetuses:

no effects observed

Description (incidence and severity):

none reported - - see Table S10 in 'Any other information on results incl tables'

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

none reported

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There was no difference in number of mated to number of pregnant females when compared to control group - see Table S10 in 'Any other information on results incl tables' for more details.

Other effects:

no effects observed

Description (incidence and severity):

no other effects reported

Details on maternal toxic effects:

maternal toxicity is noted

Key result

Dose descriptor:

NOAEL

Effect level:

36 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

clinical signs

histopathology: non-neoplastic

pre and post implantation loss

Abnormalities:

effects observed, treatment-related

Localisation:

uterus

Description (incidence and severity):

increase in Post-implantation Loss.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 330 mg/kg bw/day dose group, body weight of male and female fetuses as well as mean fetuses weight of litter was significantly decreased compared to the control vehicle group (respectively, by 11.2 %, 11.8 %, and 11.5 %). See Table S14 in in 'Any other information on results incl tables' for more details.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

See Table S10 in in 'Any other information on results incl tables' for more details.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The fetuses sex ratio in all dose treated groups was not significantly changed
compared to the control vehicle group.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

In the 330 mg/kg bw/day dose group, body weight of male and female fetuses as well as mean
fetuses weight of litter was significantly decreased compared to the control vehicle group
(respectively, by 11.2 %, 11.8 %, and 11.5 %).

Anogenital distance of all rodent fetuses:

effects observed, treatment-related

Description (incidence and severity):

The absolute and normalized anogenital distance was not changed in male fetuses of all dose test item groups. For female fetuses, the normalized anogenital distance was statistically increased in the 330 mg/kg bw/day dose group.

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Description (incidence and severity):

There were no fetuses with external abnormalities recognized as malformations in the 36 mg/kg bw/day dose group.
In the 120 mg/kg bw/day dose group, three fetuses from one litter (No.90-2m, No. 90-3m and No. 90-4m) were with inward rotated hindlimbs. No skeletal and visceral malformations were observed for these fetuses; however, the altered ossifications and soft disintegrating consistency of skeleton can be noted for No.90-2m and No. 90-3m fetuses. Besides, these fetuses had a domed head as an external observation with unknown importance. In fetus No.90-4m, dilated ventricles of the brain were noted, which can be an associated finding. In the 120 mg/kg bw/day dose group, the increase in the incidence of brain ventricles dilation was noted (Section 11.5.3) during a visceral examination. However, in the 330 mg/kg bw/day dose
group, this alteration with unknown importance was not observed. Also, in 120 mg/kg bw/day dose group, there was no significant increase in fetal and litter incidence of skeletal observations, so these findings in one litter can be considered as with unclear relationship with the test item.
In the 330 mg/kg bw/day dose group, one fetus (52-7f) was with following malformations:
inward rotated hindlimbs, absent of some digits on the left hindlimb, thread-like tail, and absent of anus (confirmed during evisceration). This fetus also had a large abdomen, probably as a result of the absence of the anus. During evisceration, no other malformations of soft tissues were found. The external malformations in the fetus No. 52-7f were associated with the skeletal malformations (see Section 11.5.4), so the treatment relation of this finding cannot be excluded despite its uniqueness.
For the fetuses in 330 mg/kg bw/day dose group, an increase of small fetus in fetal and litter incidence was observed. This increase was not significant (p = 0.053, Fisher Exact test), however, correlated to the decrease in fetal body weight in this group, and considered as test-item related.
See Tables S15-1, S15-2, S16-1, S17-1, S17-2 in 'Any other information on results incl tables' for details.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Skeletal malformations were found in one fetus (No.52-7f) from the 330 mg/kg bw/day group.
This fetus had no vertebrae after the thoracic T4, had only 7 ribs on the right and left, of which the 5th, 6th, and 7th pairs were unossified. During the external observation after the cesarean section, the few digits were recorded in the left hindpaw. Due to the low body weight of this fetus and soft consistency of limbs, the skin was not completely removed from the phalanges of hindlimbs to avoid damage and loss of structure during fetus processing. Phalanx cartilages of hindpaws were not stained appropriately; however, for left hindpaw, the absence of distal phalanges in 1st to 3d digits, absence of all phalanges in 4th digit, and absence of distal and medial phalanges in 5th digit are supposed; four metatarsals (Mt2-Mt5) were ossified. These
malformations were associated with external observations (thread-like tail). Despite the uniqueness of this case, it considered being test item-related.
Fetal skeletal variations which supposed to be related to the test item were observed in the 330 mg/kg bw/day dose group. The statistical significant increase in incidence. compared to the control vehicle group was noted for incomplete ossification of 2nd - 4th sternebra (fetal incidence, p < 0.05), unossification of 6th sternebra (fetal and litter incidence, p < 0.05), unossification in 5th metacarpal (fetal and litter incidence, p < 0.001 and p < 0.01) or its incomplete ossification (fetal incidence, p < 0.05).
The total affected fetuses with alteration in ossification was statistically increased for sternebra (fetal incidence, and mean of fetus per litter, p < 0.01), for metacarpal (fetal and litter incidence, and mean of fetus per litter, p < 0.01), and for total variations (fetal incidence, p < 0.001, and mean of fetus per litter, p < 0.01).
See Tables S15-1, S15-2, S17-1, S17-2 in 'Any other information on results incl tables' for details.

Visceral malformations:

no effects observed

Description (incidence and severity):

No test item related malformations of fetal soft tissues were found. Findings of unilateral absent of pair organs (eye, and testis with epididymis and vas deferens) were considered as incidental, not test item related.
The statistical increase in the incidence of variations was observed in the 330 mg/kg bw/day dose group for the liver (discoloration - pale, spotted), kidney (discolored due to the hemorrhage), and uterus (thin uterine horns). The percentage increase was observed for the fetal incidence (liver and kidney), as well as for the litter incidence (kidney, uterus, and liver (non-significant)). Discoloration of renal pelvis content had a low incidence; however, it was associated with the hemorrhage in the kidney. The slight non-statistical dose-dependence was noted for these findings, so, they considered as test item related. It should be noted that thin uterine horns are regarded as of unknown importance and may be due to the small fetuses in the high dose group, recorded during an external examination and discussed above.
Alterations of unknown importance were observed in the 120 mg/kg bw/day dose group in the brain and kidney. The fetal and litter incidence of dilated brain ventricles was statistically increased compared to the control group as well as the dilated renal pelvis. In the 330 mg/kg bw/day dose group, the dilation of the fourth brain ventricle was found in 4 fetuses from 2 litters, and the dilation of the renal pelvis was found in 1 fetus, which did not significantly higher compared to the control group. So, the dilated brain ventricles and dilated renal pelvis in the 120 mg/kg bw/day dose group are considered to be with unclear relation to the test item administrations.
Other findings in soft tissues presented in Table S16-4 were recorded with approximately equal or/and low frequency in all groups and considered to be as not treatment-related. The fetal and litter incidence of testis malposition was the same in the test item treated and control groups. In most fetuses, this observation was expressed as a unilateral undescended testis at half the distance from the kidney.
The total affected fetuses (incidence value and mean per litter value) were not significantly changed in test item treated groups compared to the control vehicle group

See Tables S15-2, S16-1, S16-2, S16-3, S16-4 in 'Any other information on results incl tables' for details..

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The fetuses sex ratio in all dose treated groups was not significantly changed compared to the control vehicle group.

Details on embryotoxic / teratogenic effects:

effects at 120 mg/kg bw/day are of unclear relation to the test item.
Therefore these effects are not considered for Effect level table below.

Key result

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

Abnormalities:

effects observed, treatment-related

Localisation:

external: anogenital distance

skeletal: sternum

other: fetus size

Description (incidence and severity):

incomplete or absent ossification in highest dose group
small fetuses in highest dose group
increased anogenital distance in female fetus in highest dose group

Developmental effects observed:

yes

Lowest effective dose / conc.:

330 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

TABLE S5 SUMMARY BODY WEIGHT AND BODY WEIGHT GAIN DATA FOR PREGNANT FEMALES

GROUP:	1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
	MEAN ± S.D.	MEAN ± S.D.	MEAN ± S.D.	MEAN ± S.D.
	N=24	N=21	N=24	N=23
Gestation day	Body weight, g
0	210 ± 14	208 ± 12	210 ± 13	210 ± 15
5	233 ± 14	231 ± 14	233 ± 15	233 ± 16
8	242 ± 15	238 ± 13	239 ± 14	234 ± 13
11	256 ± 16	254 ± 15	253 ± 14	248 ± 14
14	269 ± 15	266 ± 16	267 ± 16	263 ± 15
17	299 ± 20	294 ± 19	295 ± 19	288 ± 17
20	342 ± 25	338 ± 24	337 ± 26	325 ± 22 (a)
Period, gestation days	Body weight gain, %
5-8	3.7 ± 1.7	3.3 ± 2.0	2.7 ± 2.0	0.7 ± 2.5 (b)
5-11	9.6 ± 1.6	9.8 ± 1.9	8.8 ± 2.9	6.4 ± 2.5 (b)
5-14	15.3 ± 2.1	15.3 ± 2.5	14.8 ± 3.2	12.2 ± 4.0 (b)
5-17	27.9 ± 4.3	27.3 ± 2.7	26.7 ± 4.3	22.6 ± 6.3 (b)
5-20	46.5 ± 6.3	46.2 ± 4.0	44.6 ± 7.1	37.5 ± 10.9 (b)

(a) = Significantly different from control group 1 at 0.05 using repeated measures ANOVA Duncan test;

(b) = Significantly different from groups 1, 2 and 3  at 0.01 using repeated measures ANOVA Duncan test.

 

TABLE S6. SUMMARY DATA OF GRAVID UTERINE WEIGHT AND FINAL MATERNAL BODY WEIGHT

GROUP:	1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
	MEAN ± S.D.	MEAN ± S.D.	MEAN ± S.D.	MEAN ± S.D.
	N=24	N=21	N=24	N=23
Body weight at necropsy on gestation day 20, g	339 ± 26	334 ± 24	334 ± 27	323 ± 22 (b)
Gravid uterine weight, g	71.2 ± 14.5	69.8 ± 10.0	68.2 ± 11.8	59.8 ± 15.1 (a)
Final body weight without uterus, g	267 ± 17	265 ± 19	265 ± 18	263 ± 16

 

 

(a) = Significantly different from control group 1 (0 mg/kg) at 0.05 using ANOVA Dunnet test;

(b) = Significantly different from control group 1 (0 mg/kg) at 0.05 using t-test.

 

 

TABLE S9. SUMMARY DATA OF CORPORA LUTEA NUMBER AND PRE-IMPLANTATION LOSS

GROUP:	1 (0 mg/kg)		2 (36 mg/kg)		3 (120 mg/kg)		4 (330 mg/kg)
Number of corpora lutea
Total, N	413		320		399		374
MEAN ± SD	17.2 ± 3.5	(N=24)	15.2 ± 2.8	(N=21)	16.6 ± 2.8	(N=24)	16.3 ± 3.5	(N=23)
Number of implantation sites
Total, N	320		283		326		300
MEAN ± SD	13.3 ± 2.8	(N=24)	13.5 ± 2.1	(N=21)	13.6 ± 2.3	(N=24)	13.0 ± 2.5	(N=23)
Pre-implantation loss
Total, N	93		37		73		74
MEAN ± SD	3.9 ± 3.3	(N=24)	1.8 ± 1.6	(N=21)	3.0 ± 2.0	(N=24)	3.2 ± 3.9	(N=23)
% of corpora lutea	22.5%		11.6%		18.3%		19.8%

 

 

 

 

TABLE S10. SUMMARY DATA OF UTERINE CONTENT

	GROUP:	1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
Number of treated females with confirmed mating	N	25	24	24	26
Number of pregnant females	N	24	21	24	23
Implantation sites	N	320	283	326	300
No. per animal	MEAN ± SD	13.3 ± 2.8	13.5 ± 2.1	13.6 ± 2.3	13.0 ± 2.5
Resorptions - Early	N	7	5	21	18
% of implantation sites		2.2	1.8	6.4 (a)	6.0 (b)
No. per animal	MEAN ± SD	0.3 ± 0.5	0.2 ± 0.5	0.9 ± 1.0 (c)	0.8 ± 1.3
Resorptions - Late	N	2	1	3	1
% of implantation sites		0.6	0.4	0.9	0.4
No. per animal	MEAN ± SD	0.1 ± 0.3	0.0 ± 0.2	0.1 ± 0.4	0.0 ± 0.2
Post-implantation Loss	Total	9	6	24	19
% of implantation sites		2.8	2.1	7.4 (a)	6.3 (b)
No. per animal	MEAN ± SD	0.4 ± 0.6	0.3 ± 0.6	1.0 ± 1.1 (c)	0.8 ± 1.3
Number of females with post-implantation loss	N / N pregnant	8 / 24	5 / 21	13/24	11 / 23
Total Fetuses	N	311	277	302	281
Alive	%	100.0	100.0	100.0	100.0
Dead	%	0.0	0.0	0.0	0.0
No. per animal	MEAN ± SD	13.0 ± 2.7	13.2 ± 2.1	12.6 ± 2.7	12.2 ± 3.2

(a) Difference from control group 0 mg/kg/day with p < 0.01, Chi-square test;

(b) Difference from control group 0 mg/kg/day with p < 0.05, Chi-square test;

(c) Difference from control group 0 mg/kg/day with p < 0.05, Mann-Whitney test.

 

 

 

TABLE S11. SUMMARY DATA OF THYROIDS WEIGHT

GROUP:	1 (0 mg/kg)		2 (36 mg/kg)		3 (120 mg/kg)		4 (330 mg/kg)
	MEAN ± S.D.	N	MEAN ± S.D.	N	MEAN ± S.D.	N	MEAN ± S.D.	N
Gravid body weight (a), g	339 ± 26	24	334 ± 24	21	334 ± 27	24	323 ± 22	23
Body weight without uterus (b), g	267 ± 17	24	265 ± 19	21	265 ± 18	24	263 ± 16	23
Thyroid weight
Absolute weight, g	0.0182 ± 0.0038	24	0.0172 ± 0.0034	21	0.0173 ± 0.0026	24	0.0171 ± 0.0023	23
Relative gravid body weight (a), g/100 g	0.0054 ± 0.0011	24	0.0052 ± 0.0009	21	0.0052 ± 0.0008	24	0.0053 ± 0.0007	23
Relative body weight (b), g/100 g	0.0068 ± 0.0013	24	0.0065 ± 0.0012	21	0.0065 ± 0.0009	24	0.0065 ± 0.0008	23

(a) Body weight of pregnant female at necropsy;

(b) Body weight without gravid uterine weight.

 

 

 

TABLE S12. SUMMARY DATA OF MICROSCOPIC FINDINGS IN THYROID GLANDS

GROUP:	1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
	N=24	N=21	N=24	N=23
Finding, N
C-cell hyperplasia	0	0	3	4 (a)
Minimal	0	0	3	4 (a)
Cyst, Congenital	6	7	10	9
Minimal	3	2	7	5
Slight	3	5	3	4
Ectopic Lymphoid tissue	2	2	2	1
Minimal	2	1	1	0
Slight	0	1	1	1
Follicular cell vacuolization, single follicle	0	0	1	0
Minimal	0	0	1	0

(a) Difference from control group 0 mg/kg/day with p < 0.05, Fisher Exact test

 

 

 

TABLE S13. SUMMARY DATA OF THYROID HORMONS SERUM LEVEL IN FEMALES

GROUP:	1 (0 mg/kg)		2 (36 mg/kg)		3 (120 mg/kg)			4 (330 mg/kg)
	MEAN ± S.D.	N	MEAN ± S.D.	N	MEAN ± S.D.	N		MEAN ± S.D.	N
Triiodothyronine (T3), ng/mL	4.587 ± 1.284	24	4.897 ± 1.611	21	4.335 ± 1.394		24	4.321 ± 1.147	23
Thyroxine (T4), ng/mL	21.97 ± 6.06	24	19.15 ± 10.16	21	21.83 ± 8.10		24	25.16 ± 9.04	23
Thyroid Stimulating Hormone (TSH), pg/mL	472.2 ± 195.9	24	451.9 ± 167.2	20	442.5 ± 217.3		24	435.5 ± 194.8	23

 

TABLE S14. SUMMARY RESULTS OF BODY WEIGHTS, ANOGENITAL DISTANCES AND SEX OF FETUSES

GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
Number animals with fetuses		N=24	N=21	N=24	N=23
Male Fetuses
Body weight, g	MEAN ± SD	3.74 ± 0.30	3.70 ± 0.20	3.82 ± 0.57	3.32 ± 0.30 (a)
AGD, mm	MEAN ± SD	2.61 ± 0.12	2.60 ± 0.12	2.61 ± 0.16	2.50 ± 0.20
AGD normalized	MEAN ± SD	1.68 ± 0.07	1.68 ± 0.08	1.68 ± 0.11	1.67 ± 0.11
Number, total	N	173	131	150	144
No. per animal	MEAN ± SD	7.2 ± 1.8	6.2 ± 1.9	6.3 ± 1.8	6.3 ± 2.2
% males	%	55.6	47.3	49.7	51.2
Female Fetuses
Body weight, g	MEAN ± SD	3.56 ± 0.26	3.50 ± 0.14	3.65 ± 0.52	3.14 ± 0.29 (a)
AGD, mm	MEAN ± SD	1.07 ± 0.08	1.08 ± 0.07	1.08 ± 0.10	1.08 ± 0.08
AGD normalized	MEAN ± SD	0.70 ± 0.05	0.71 ± 0.05	0.70 ± 0.07	0.74 ± 0.06 (b)
Number, total	N	138	146	152	137
No. per animal	MEAN ± SD	5.8 ± 2.4	7.0 ± 1.7	6.3 ± 2.3	6.0 ± 2.5
% females	%	44.4	52.7	50.3	48.8
Mean fetuses body weight, g	MEAN ± SD	3.66 ± 0.28	3.59 ± 0.16	3.74 ± 0.54	3.24 ± 0.27 (c)

(a) Significantly different from control group 1 at 0.001 using ANOVA Dunnett test;

(b) Difference from control group 0 mg/kg/day with p < 0.05, ANOVA Kruscal-Wallis test;

(c) Significantly different from control group 1 at 0.001 using ANOVA covariant test with litter size as a covariant.

 

 

TABLE S15-1. SUMMARY RESULTS OF FETUSES EXTERNAL MALFORMATIONS DATA

GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
EXTERNAL MALFORMATIONS
Litters Evaluated	N	24	21	24	23
Fetuses Evaluated		311	277	302	281
Live	N	311	277	302	281
Dead	N	0	0	0	0
HINDLIMBS
Malrotated, inward
Fetal Incidence	N	0	0	3	1
	%	0.0	0.0	1.0	0.4
Litter Incidence	N	0	0	1	1
	%	0.0	0.0	4.2	4.3
Paw, Digits – Few
Fetal Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	0.4
Litter Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	4.3
TAIL – Thread-like
Fetal Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	0.4
Litter Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	4.3
TRUNK
Anus - Absent
Fetal Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	0.4
Litter Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	4.3
TOTAL AFFECTED FETUSES
Fetal Incidence	N	0	0	3	1
	%	0.0	0.0	1.0	0.4
Litter Incidence	N	0	0	1	1
	%	0.0	0.0	4.2	4.3
Mean per litter, %	MEAN ± SD	0.00 ± 0.00	0.00 ± 0.00	1.1 ± 5.6	0.3 ± 1.6

 

 

 

 

TABLE S15-2. SUMMARY RESULTS OF FETUSES VARIATIONS AND OTHER OBSERVATION DATA

GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
EXTERNAL VARIATIONS AND OTHER OBSERVATIONS
Litters Evaluated	N	24	21	24	23
Fetuses Evaluated		311	277	302	281
Live	N	311	277	302	281
Dead	N	0	0	0	0
GENERAL
Subcutaneous hemorrhage
Fetal Incidence	N	58	41	52	42
	%	18.6	14.8	17.2	14.9
Litter Incidence	N	17	19	16	18
	%	70.8	90.5	66.7	78.3
Intraperitoneal hemorrhage
Fetal Incidence	N	0	1	0	0
	%	0.0	0.4	0.0	0.0
Litter Incidence	N	0	1	0	0
	%	0.0	4.8	0.0	0.0
Fetus - Small
Fetal Incidence	N	2	0	3	8
	%	0.6	0.0	1.0	2.8
Litter Incidence	N	2	0	3	4
	%	8.3	0.0	12.5	17.4
Fetus - Pale
Fetal Incidence	N	1	0	0	1
	%	0.3	0.0	0.0	0.4
Litter Incidence	N	1	0	0	1
	%	4.2	0.0	0.0	4.3
HEAD/NECK
Domed head
Fetal Incidence	N	0	0	3	0
	%	0.0	0.0	1.0	0.0
Litter Incidence	N	0	0	1	0
	%	0.0	0.0	4.2	0.0
TRUNK	%
Genital Tubercle- Large
Fetal Incidence	N	6	2	1	6
	%	1.9	0.7	0.3	2.1
Litter Incidence	N	2	2	1	3
	%	8.3	9.5	4.2	13.0
Abdomen - Enlarged
Fetal Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	0.4
Litter Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	4.3
TOTAL AFFECTED FETUSES
Fetal Incidence	N	64	44	54	53
	%	20.6	15.9	17.9	18.9
Litter Incidence	N	19	20	17	20
	%	79.2	95.2	70.8	87.0
Mean per litter, %	MEAN ± SD	19.3 ± 15.9	15.7 ± 12.4	16.5 ± 14.3	19.6 ± 13.5

 

 

 

TABLE S16-1. SUMMARY DATA OF FETAL SOFT TISSUE MALFORMATIONS

GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
Litters Evaluated	N	24	21	24	23
Fetuses Evaluated	N	153	138	149	139
Males	N	89	67	81	75
Females	N	64	71	68	64
EYE – ABSENT (unilateral)
Fetal Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	0.7
Litter Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	4.3
Affected fetuses per Litter	MEAN%	0.0	0.0	0.0	0.6
	SD	0.0	0.0	0.0	3.0
TESTIS – ABSENT (unilateral)
Fetal Incidence	N	1	0	1	0
	%	1.1	0.0	1.2	0.0
Litter Incidence	N	1	0	1	0
	%	4.2	0.0	4.2	0.0
Affected fetuses per Litter	MEAN%	0.6	0.0	1.0	0.0
	SD	2.9	0.0	5.1	0.0
EPIDIDYMIS – ABSENT (unilateral)
Fetal Incidence	N	1	0	1	0
	%	1.1	0.0	1.2	0.0
Litter Incidence	N	1	0	1	0
	%	4.2	0.0	4.2	0.0
Affected fetuses per Litter	MEAN%	0.6	0.0	1.0	0.0
	SD	2.9	0.0	5.1	0.0
VAS DEFERENS – ABSENT (unilateral)
Fetal Incidence	N	1	0	1	0
	%	1.1	0.0	1.2	0.0
Litter Incidence	N	1	0	1	0
	%	4.2	0.0	4.2	0.0
Affected fetuses per Litter	MEAN%	0.6	0.0	1.0	0.0
	SD	2.9	0.0	5.1	0.0

 

 

 

TABLE S16-2. SUMMARY DATA OF FETAL SOFT TISSUE VARIATIONS AND OTHER ALTERATIONS: FINDINGS ARE CONSIDERED TO BE ASSOCIATED WITH ADMINISTRATION OF THE TEST ITEM

	GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
	Litters Evaluated	N	24	21	24	23
	Fetuses Evaluated	N	153	138	149	139
	Males	N	89	67	81	75
	Females	N	64	71	68	64
	LIVER
LIVER LOBE - Discolored (pale, spots)
Fetal Incidence		N	2	2	4	12
		%	1.3	1.4	2.7	8.6 (a)
Litter Incidence		N	2	2	3	6
		%	8.3	9.5	12.5	26.1
Affected fetuses per Litter		MEAN%	1.4	1.4	2.9	7.3
		SD	4.7	4.3	8.9	18.5
	KIDNEY
KIDNEY - Discolored (hemorrhage)
Fetal Incidence		N	1	0	4	9
		%	0.7	0.0	2.7	6.5 (a)
Litter Incidence		N	1	0	4	6
		%	4.2	0.0	16.7	26.1 (b)
Affected fetuses per Litter		MEAN%	0.5	0.0	2.5	7.0
		SD	2.6	0.0	5.9	13.9
RENAL PELVIS CONTENT - Discolored
Fetal Incidence		N	0	0	0	2
		%	0.0	0.0	0.0	1.4
Litter Incidence		N	0	0	0	2
		%	0.0	0.0	0.0	8.7
Affected fetuses per Litter		MEAN%	0.0	0.0	0.0	2.8
		SD	0.0	0.0	0.0	10.7
	UTERUS
UTERINE HORNS - Thin
Fetal Incidence		N	0	2	2	4
		%	0.0	2.8	2.9	6.3
Litter Incidence		N	0	2	2	4
		%	0.0	9.5	8.3	17.4 (b)
Affected fetuses per Litter		MEAN%	0.0	3.6	2.4	6.5
		SD	0.0	12.0	8.3	15.5

(a) Significantly different from control group 1 at 0.01 using Fisher Exact test;

(b) Significantly different from control group 1 at 0.05 using Fisher Exact test

 

 

 

TABLE S16-3. SUMMARY DATA OF FETAL SOFT TISSUE VARIATIONS AND OTHER ALTERATIONS: FINDINGS OF UNCLEAR RELATION TO THE TEST ITEM ADMINISTRATION

	GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
	Litters Evaluated	N	24	21	24	23
	Fetuses Evaluated	N	153	138	149	139
	Males	N	89	67	81	75
	Females	N	64	71	68	64
	BRAIN
	FOURTH VENTRICLE - Dilated
	Fetal Incidence	N	0	0	8	0
		%	0.0	0.0	5.4 (a)	0.0
	Litter Incidence	N	0	0	4	0
		%	0.0	0.0	16.7 (b)	0.0
	Affected fetuses per Litter	MEAN%	0.0	0.0	4.8	0.0
		SD	0.0	0.0	12.4	0.0
	LATERAL VENTRICLE - Dilated
	Fetal Incidence	N	1	0	8	4
		%	0.7	0.0	5.4 (b)	2.9
	Litter Incidence	N	1	0	4	2
		%	4.2	0.0	16.7	8.7
	Affected fetuses per Litter	MEAN%	0.5	0.0	5.0	3.3
		SD	2.6	0.0	15.2	11.1
	THIRD VENTRICLE - Dilated
	Fetal Incidence	N	0	0	5	0
		%	0.0	0.0	3.4 (b)	0.0
	Litter Incidence	N	0	0	4	0
		%	0.0	0.0	16.7 (b)	0.0
	Affected fetuses per Litter	MEAN%	0.0	0.0	3.2	0.0
		SD	0.0	0.0	7.7	0.0
	VENTRICLE - Dilated
	Fetal Incidence	N	0	0	5	0
		%	0.0	0.0	3.4 (a)	0.0
	Litter Incidence	N	0	0	5	0
		%	0.0	0.0	20.8 (a)	0.0
	Affected fetuses per Litter	MEAN%	0.0	0.0	3.4	0.0
		SD	0.0	0.0	7.2	0.0
	KIDNEY
RENAL PELVIS - Dilated
Fetal Incidence		N	2	6	10	1
		%	1.3	4.3	6.7 (b)	0.7
Litter Incidence		N	2	4	8	1
		%	8.3	19.0	33.3 (a)	4.3
Affected fetuses per Litter		MEAN%	1.5	4.9	7.0	0.6
		SD	5.2	13.7	12.1	3.0

(a) Significantly different from control group 1 at 0.01 using Fisher Exact test;

(b) Significantly different from control group 1 at 0.05 using Fisher Exact test

 

 

 

 

TABLE S16-4. SUMMARY DATA OF FETAL SOFT TISSUE VARIATIONS AND OTHER ALTERATIONS: FINDINGS WERE NOT CONSIDERED TO BE ASSOCIATED WITH ADMINISTRATION OF THE TEST ITEM

GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
Litters Evaluated	N	24	21	24	23
Fetuses Evaluated	N	153	138	149	139
Males	N	89	67	81	75
Females	N	64	71	68	64
ABDOMEN-Intra-abdominal hemorrhage
Fetal Incidence	N	7	13	11	7
	%	4.6	9.4	7.4	5.0
Litter Incidence	N	5	7	3	7
	%	20.8	33.3	12.5	30.4
Affected fetuses per Litter	MEAN%	4.0	9.1	8.5	6.3
	SD	9.2	14.3	23.7	12.0
BRAIN
CEREBELLUM - Red material
Fetal Incidence	N	0	1	0	1
	%	0.0	0.7	0.0	0.7
Litter Incidence	N	0	1	0	1
	%	0.0	4.8	0.0	4.3
Affected fetuses per Litter	MEAN%	0.0	0.7	0.0	0.5
	SD	0.0	3.1	0.0	2.6
OLFACTORY LOBE - Large (unilateral)
Fetal Incidence	N	0	1	0	1
	%	0.0	0.7	0.0	0.7
Litter Incidence	N	0	1	0	1
	%	0.0	4.8	0.0	4.3
Affected fetuses per Litter	MEAN%	0.0	0.7	0.0	0.6
	SD	0.0	3.1	0.0	3.0
PERIMENINGEAL SPACE - Red material
Fetal Incidence	N	12	3	10	7
	%	7.8	2.2	6.7	5.0
Litter Incidence	N	8	3	6	4
	%	33.3	14.3	25.0	17.4
Affected fetuses per Litter	MEAN%	8.9	2.4	7.6	5.8
	SD	15.2	6.2	15.7	16.7
EAR, Inner/Middle - Red material
Fetal Incidence	N	3	4	5	3
	%	2.0	2.9	3.4	2.2
Litter Incidence	N	3	4	5	3
	%	12.5	19.0	20.8	13.0
Affected fetuses per Litter	MEAN%	1.8	2.9	3.6	2.0
	SD	4.9	6.1	7.3	5.2

 

 

 

 

TABLE S16-4. SUMMARY DATA OF FETAL SOFT TISSUE VARIATIONS AND OTHER ALTERATIONS: FINDINGS WERE NOT CONSIDERED TO BE ASSOCIATED WITH ADMINISTRATION OF THE TEST ITEM, CONTINUATION

GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
Litters Evaluated	N	24	21	24	23
Fetuses Evaluated	N	153	138	149	139
Males	N	89	67	81	75
Females	N	64	71	68	64
HEART
ATRIUM - Discolored (pale)
Fetal Incidence	N	0	1	0	1
	%	0.0	0.7	0.0	0.7
Litter Incidence	N	0	1	0	1
	%	0.0	4.8	0.0	4.3
Affected fetuses per Litter	MEAN%	0.0	0.7	0.0	0.5
	SD	0.0	3.1	0.0	2.6
ATRIUM - Large
Fetal Incidence	N	0	1	0	0
	%	0.0	0.7	0.0	0.0
Litter Incidence	N	0	1	0	0
	%	0.0	4.8	0.0	0.0
Affected fetuses per Litter	MEAN%	0.0	0.7	0.0	0.0
	SD	0.0	3.1	0.0	0.0
ATRIUM - Small
Fetal Incidence	N	0	0	1	0
	%	0.0	0.0	0.7	0.0
Litter Incidence	N	0	0	1	0
	%	0.0	0.0	4.2	0.0
Affected fetuses per Litter	MEAN%	0.0	0.0	0.8	0.0
	SD	0.0	0.0	4.1	0.0
PERICARDIUM - Blood filled
Fetal Incidence	N	4	0	3	0
	%	2.6	0.0	2.0	0.0
Litter Incidence	N	1	0	3	0
	%	4.2	0.0	12.5	0.0
Affected fetuses per Litter	MEAN%	2.8	0.0	2.1	0.0
	SD	13.6	0.0	5.8	0.0
VENTRICLES - Dilated, Blood-filled
Fetal Incidence	N	15	10	9	11
	%	9.8	7.2	6.0	7.9
Litter Incidence	N	4	6	5	4
	%	16.7	28.6	20.8	17.4
Affected fetuses per Litter	MEAN%	9.6	7.6	5.5	6.9
	SD	25.0	13.9	13.8	19.0
VENTRICLE WALL - Discolored
Fetal Incidence	N	0	0	3	0
	%	0.0	0.0	2.0	0.0
Litter Incidence	N	0	0	1	0
	%	0.0	0.0	4.2	0.0
Affected fetuses per Litter	MEAN%	0.0	0.0	2.5	0.0
	SD	0.0	0.0	12.2	0.0

 

 

 

TABLE S16-4. SUMMARY DATA OF FETAL SOFT TISSUE VARIATIONS AND OTHER ALTERATIONS: FINDINGS WERE NOT CONSIDERED TO BE ASSOCIATED WITH ADMINISTRATION OF THE TEST ITEM, CONTINUATION

GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
Litters Evaluated	N	24	21	24	23
Fetuses Evaluated	N	153	138	149	139
Males	N	89	67	81	75
Females	N	64	71	68	64
AORTA - Dilated
Fetal Incidence	N	0	1	0	0
	%	0.0	0.7	0.0	0.0
Litter Incidence	N	0	1	0	0
	%	0.0	4.8	0.0	0.0
Affected fetuses per Litter	MEAN%	0.0	0.7	0.0	0.0
	SD	0.0	3.1	0.0	0.0
CAROTID ARTERY - Dilated
Fetal Incidence	N	0	0	1	0
	%	0.0	0.0	0.7	0.0
Litter Incidence	N	0	0	1	0
	%	0.0	0.0	4.2	0.0
Affected fetuses per Litter	MEAN%	0.0	0.0	0.6	0.0
	SD	0.0	0.0	2.9	0.0
VEIN, HEPATIC - Dilated
Fetal Incidence	N	5	3	3	7
	%	3.3	2.2	2.0	5.0
Litter Incidence	N	3	1	2	4
	%	12.5	4.8	8.3	17.4
Affected fetuses per Litter	MEAN%	3.3	2.0	2.0	4.4
	SD	9.6	9.4	7.0	8.8
VEIN, CAUDAL - Dilated
Fetal Incidence	N	0	1	1	0
	%	0.0	0.7	0.7	0.0
Litter Incidence	N	0	1	1	0
	%	0.0	4.8	4.2	0.0
Affected fetuses per Litter	MEAN%	0.0	1.0	0.6	0.0
	SD	0.0	4.4	2.9	0.0
VEIN, RENAL - Dilated
Fetal Incidence	N	3	0	2	5
	%	2.0	0.0	1.3	3.6
Litter Incidence	N	2	0	1	2
	%	8.3	0.0	4.2	8.7
Affected fetuses per Litter	MEAN%	1.9	0.0	1.7	4.1
	SD	7.2	0.0	8.2	13.6

 

 

 

TABLE S16-4. SUMMARY DATA OF FETAL SOFT TISSUE VARIATIONS AND OTHER ALTERATIONS: FINDINGS WERE NOT CONSIDERED TO BE ASSOCIATED WITH ADMINISTRATION OF THE TEST ITEM, CONTINUATION

GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
Litters Evaluated	N	24	21	24	23
Fetuses Evaluated	N	153	138	149	139
Males	N	89	67	81	75
Females	N	64	71	68	64
LIVER
LIVER LOBE - Discolored (hemorrhage)
Fetal Incidence	N	5	3	4	2
	%	3.3	2.2	2.7	1.4
Litter Incidence	N	5	3	2	2
	%	20.8	14.3	8.3	8.7
Affected fetuses per Litter	MEAN%	3.1	2.1	2.9	1.4
	SD	6.2	5.2	9.8	4.8
LIVER LOBE, Vessels - Dilated
Fetal Incidence	N	1	0	1	0
	%	0.7	0.0	0.7	0.0
Litter Incidence	N	1	0	1	0
	%	4.2	0.0	4.2	0.0
Affected fetuses per Litter	MEAN%	0.7	0.0	0.6	0.0
	SD	3.4	0.0	2.9	0.0
BILE DUCT - Dilated
Fetal Incidence	N	0	0	1	1
	%	0.0	0.0	0.7	0.7
Litter Incidence	N	0	0	1	1
	%	0.0	0.0	4.2	4.3
Affected fetuses per Litter	MEAN%	0.0	0.0	0.6	0.5
	SD	0.0	0.0	2.9	2.3
STOMACH, WALL - Alteration of texture
Fetal Incidence	N	0	0	4	0
	%	0.0	0.0	2.7	0.0
Litter Incidence	N	0	0	1	0
	%	0.0	0.0	4.2	0.0
Affected fetuses per Litter	MEAN%	0.0	0.0	3.3	0.0
	SD	0.0	0.0	16.3	0.0
STOMACH, WALL - Discoloration
Fetal Incidence	N	0	0	3	0
	%	0.0	0.0	2.0	0.0
Litter Incidence	N	0	0	1	0
	%	0.0	0.0	4.2	0.0
Affected fetuses per Litter	MEAN%	0.0	0.0	2.5	0.0
	SD	0.0	0.0	12.2	0.0
PANCREAS - Discolored (dark)
Fetal Incidence	N	1	1	0	1
	%	0.7	0.7	0.0	0.7
Litter Incidence	N	1	1	0	1
	%	4.2	4.8	0.0	4.3
Affected fetuses per Litter	MEAN%	0.5	0.6	0.0	0.5
	SD	2.6	2.7	0.0	2.6

 

 

 

TABLE S16-4. SUMMARY DATA OF FETAL SOFT TISSUE VARIATIONS AND OTHER ALTERATIONS: FINDINGS WERE NOT CONSIDERED TO BE ASSOCIATED WITH ADMINISTRATION OF THE TEST ITEM, CONTINUATION

GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
Litters Evaluated	N	24	21	24	23
Fetuses Evaluated	N	153	138	149	139
Males	N	89	67	81	75
Females	N	64	71	68	64
SPLEEN - Discolored (dark)
Fetal Incidence	N	1	0	0	1
	%	0.7	0.0	0.0	0.7
Litter Incidence	N	1	0	0	1
	%	4.2	0.0	0.0	4.3
Affected fetuses per Litter	MEAN%	0.5	0.0	0.0	0.5
	SD	2.6	0.0	0.0	2.6
INTESTINE WALL - Discolored (dark)
Fetal Incidence	N	0	2	4	0
	%	0.0	1.4	2.7	0.0
Litter Incidence	N	0	2	2	0
	%	0.0	9.5	8.3	0.0
Affected fetuses per Litter	MEAN%	0.0	1.3	3.1	0.0
	SD	0.0	4.0	12.5	0.0
INTESTINE WALL - Alteration of texture
Fetal Incidence	N	0	0	3	0
	%	0.0	0.0	2.0	0.0
Litter Incidence	N	0	0	1	0
	%	0.0	0.0	4.2	0.0
Affected fetuses per Litter	MEAN%	0.0	0.0	2.5	0.0
	SD	0.0	0.0	12.2	0.0
KIDNEY
RENAL PAPILLA - Small
Fetal Incidence	N	2	1	4	1
	%	1.3	0.7	2.7	0.7
Litter Incidence	N	2	1	4	1
	%	8.3	4.8	16.7	4.3
Affected fetuses per Litter	MEAN%	1.5	0.7	2.6	0.6
	SD	5.2	3.1	6.2	3.0
RENAL PELVIS - Small
Fetal Incidence	N	2	0	0	0
	%	1.3	0.0	0.0	0.0
Litter Incidence	N	2	0	0	0
	%	8.3	0.0	0.0	0.0
Affected fetuses per Litter	MEAN%	1.2	0.0	0.0	0.0
	SD	5.8	0.0	0.0	0.0

 

 

 

TABLE S16-4. SUMMARY DATA OF FETAL SOFT TISSUE VARIATIONS AND OTHER ALTERATIONS: FINDINGS WERE NOT CONSIDERED TO BE ASSOCIATED WITH ADMINISTRATION OF THE TEST ITEM, CONTINUATION

GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
Litters Evaluated	N	24	21	24	23
Fetuses Evaluated	N	153	138	149	139
Males	N	89	67	81	75
Females	N	64	71	68	64
KIDNEY
KIDNEY - Malpositioned
Fetal Incidence	N	0	1	1	2
	%	0.0	0.7	0.7	1.4
Litter Incidence	N	0	1	1	2
	%	0.0	4.8	4.2	8.7
Affected fetuses per Litter	MEAN%	0.0	0.8	0.6	1.0
	SD	0.0	3.6	2.9	3.4
KIDNEY - Altered texture
Fetal Incidence	N	0	1	1	0
	%	0.0	0.7	0.7	0.0
Litter Incidence	N	0	1	1	0
	%	0.0	4.8	4.2	0.0
Affected fetuses per Litter	MEAN%	0.0	0.6	0.5	0.0
	SD	0.0	2.7	2.3	0.0
KIDNEY - Large
Fetal Incidence	N	0	1	0	0
	%	0.0	0.7	0.0	0.0
Litter Incidence	N	0	1	0	0
	%	0.0	4.8	0.0	0.0
Affected fetuses per Litter	MEAN%	0.0	0.8	0.0	0.0
	SD	0.0	3.6	0.0	0.0
URINARY BLADDER
URINARY BLADDER - Discolored (dark)
Fetal Incidence	N	0	2	0	0
	%	0.0	1.4	0.0	0.0
Litter Incidence	N	0	2	0	0
	%	0.0	9.5	0.0	0.0
Affected fetuses per Litter	MEAN%	0.0	1.3	0.0	0.0
	SD	0.0	4.0	0.0	0.0
URINARY BLADDER - Small
Fetal Incidence	N	0	1	0	0
	%	0.0	0.7	0.0	0.0
Litter Incidence	N	0	1	0	0
	%	0.0	4.8	0.0	0.0
Affected fetuses per Litter	MEAN%	0.0	0.8	0.0	0.0
	SD	0.0	3.6	0.0	0.0

 

 

 

TABLE S16-4. SUMMARY DATA OF FETAL SOFT TISSUE VARIATIONS AND OTHER ALTERATIONS: FINDINGS WERE NOT CONSIDERED TO BE ASSOCIATED WITH ADMINISTRATION OF THE TEST ITEM, CONTINUATION

GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
Litters Evaluated	N	24	21	24	23
Fetuses Evaluated	N	153	138	149	139
Males	N	89	67	81	75
Females	N	64	71	68	64
TESTIS - Malpositioned
Fetal Incidence	N	5	5	4	6
	%	5.6	7.5	4.9	8.0
Litter Incidence	N	5	4	4	5
	%	20.8	19.0	16.7	21.7
Affected fetuses per Litter	MEAN%	5.3	7.9	6.3	10.4
	SD	10.8	18.5	15.2	24.6
VAS DEFERENS - Long
Fetal Incidence	N	2	1	0	0
	%	2.2	1.5	0.0	0.0
Litter Incidence	N	2	1	0	0
	%	8.3	4.8	0.0	0.0
Affected fetuses per Litter	MEAN%	2.2	1.2	0.0	0.0
	SD	7.8	5.5	0.0	0.0
OVARIES - Small
Fetal Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	1.6
Litter Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	4.3
Affected fetuses per Litter	MEAN%	0.0	0.0	0.0	2.2
	SD	0.0	0.0	0.0	10.4
UTERUS
UTERUS - Discolored (dark)
Fetal Incidence	N	0	2	0	0
	%	0.0	2.8	0.0	0.0
Litter Incidence	N	0	2	0	0
	%	0.0	9.5	0.0	0.0
Affected fetuses per Litter	MEAN%	0.0	2.8	0.0	0.0
	SD	0.0	8.9	0.0	0.0
UTERINE HORNS - Short
Fetal Incidence	N	0	1	0	0
	%	0.0	1.4	0.0	0.0
Litter Incidence	N	0	1	0	0
	%	0.0	4.8	0.0	0.0
Affected fetuses per Litter	MEAN%	0.0	1.6	0.0	0.0
	SD	0.0	7.3	0.0	0.0
TOTAL AFFECTED FETUSES
Fetal Incidence	N	45	40	52	46
	%	29.4	29.0	34.9	33.1
Litter Incidence	N	18	17	18	21
	%	75.0	81.0	75.0	91.3
Affected fetuses per Litter	MEAN%	29.9	29.5	34.4	34.5
	SD	29.7	24.7	29.9	31.4

 

 

 

TABLE S17-1. SUMMARY DATA OF FETAL SKELETAL MALFORMATIONS

GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
Litters Evaluated	N	24	21	24	23
Fetuses Evaluated	N	156	139	153	142
VERTEBRAE - Absent
Fetal Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	0.7
Litter Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	4.3
Affected fetuses per Litter	MEAN%	0.0	0.0	0.0	0.6
	SD	0.0	0.0	0.0	3.0
RIBS - Absent
Fetal Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	0.7
Litter Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	4.3
Affected fetuses per Litter	MEAN%	0.0	0.0	0.0	0.6
	SD	0.0	0.0	0.0	3.0
HINDPAW PHALANX - Absent
Fetal Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	0.7
Litter Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	4.3
Affected fetuses per Litter	MEAN%	0.0	0.0	0.0	0.6
	SD	0.0	0.0	0.0	3.0

 

 

 

TABLE S17-2. SUMMARY DATA OF FETAL SKELETAL AND CARTILAGE VARIATIONS

GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
Litters Evaluated	N	24	21	24	23
Fetuses Evaluated	N	156	139	153	142
STERNEBRA
Incomplete ossification of 2nd to 4th sternebra
Fetal Incidence	N	19	7	11	30
	%	12.2	5.0	7.2	21.1 (b)
Litter Incidence	N	11	11	11	16
	%	45.8	52.4	45.8	69.6
Unossified 6th sternebra
Fetal Incidence	N	12	2	10	23
	%	7.7	1.4	6.5	16.2 (b)
Litter Incidence	N	5	5	5	13
	%	20.8	23.8	20.8	56.5 (b)
Incomplete ossification in 5th sternebra
Fetal Incidence	N	19	9	22	18
	%	12.2	6.5	14.4	12.7
Litter Incidence	N	9	9	9	11
	%	37.5	42.9	37.5	47.8
Incomplete ossification in 6th sternebra
Fetal Incidence	N	33	23	26	42
	%	21.2	16.5	17.0	29.6
Litter Incidence	N	13	13	13	18
	%	54.2	61.9	54.2	78.3
TOTAL AFFECTED IN STERNEBRA
Fetal Incidence	N	57	28	46	76
	%	36.5	20.1	30.1	53.5 (a)
Litter Incidence	N	17	17	17	20
	%	70.8	81.0	70.8	87.0
Affected fetuses per Litter	MEAN%	34.1	20.8	29.6	57.5 (c)
	SD	29.2	25.8	25.8	34.6

(a) Significantly different from control group 1 at 0.01 using Fisher Exact  test;

(b) Significantly different from control group 1 at 0.05 using Fisher Exact  test;

(c) Significantly different from control group 1 at 0.01 using Kruscal-Wallis  test

 

 

 

 

TABLE S17-2. SUMMARY DATA OF FETAL SKELETAL AND CARTILAGE VARIATIONS, CONTINUATION

GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
Litters Evaluated	N	24	21	24	23
Fetuses Evaluated	N	156	139	153	142
RIBS
Rib - Supernumerary – 14th
Fetal Incidence	N	0	0	1	0
	%	0.0	0.0	0.7	0.0
Litter Incidence	N	0	0	1	0
	%	0.0	0.0	4.2	0.0
Rib, Costal cartilage - Not articulated with sternum
Fetal Incidence	N	1	0	1	0
	%	0.6	0.0	0.7	0.0
Litter Incidence	N	1	0	1	0
	%	4.2	0.0	4.2	0.0
Ribs - Unossified
Fetal Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	0.7
Litter Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	4.3
TOTAL AFFECTED IN RIBS
Fetal Incidence	N	1	0	2	1
	%	0.6	0.0	1.3	0.7
Litter Incidence	N	1	0	2	1
	%	4.2	0.0	8.3	4.3
Affected fetuses per Litter	MEAN%	4.2	0.0	1.3	0.6
	SD	0.7	0.0	4.6	3.0
METACARPAL
Unossified 5th Metacarpal
Fetal Incidence	N	34	19	25	66
	%	21.8	13.7	16.3	46.5 (a)
Litter Incidence	N	11	11	11	21
	%	45.8	52.4	45.8	91.3 (b)
Incomplete ossification of 5th Metacarpal
Fetal Incidence	N	7	4	7	16
	%	4.5	2.9	4.6	11.3 (c)
Litter Incidence	N	6	6	6	9
	%	25.0	28.6	25.0	39.1
TOTAL AFFECTED IN METACARPAL
Fetal Incidence	N	41	23	32	83
	%	26.3	16.5	20.9	58.5 (a)
Litter Incidence	N	13	13	13	21
	%	54.2	61.9	54.2	91.3 (b)
Affected fetuses per Litter	MEAN%	23.2	15.9	19.1	60.7 (d)
	SD	29.2	17.5	27.3	34.2

(a) Significantly different from control group 1 at 0.001 using Fisher Exact  test;

(b) Significantly different from control group 1 at 0.01 using Fisher Exact  test;

(c) Significantly different from control group 1 at 0.05 using Fisher Exact  test;

(d) Significantly different from control group 1 at 0.01 using Kruscal-Wallis  test

 

 

 

TABLE S17-2. SUMMARY DATA OF FETAL SKELETAL AND CARTILAGE VARIATIONS, CONTINUATION

GROUP:		1 (0 mg/kg)	2 (36 mg/kg)	3 (120 mg/kg)	4 (330 mg/kg)
Litters Evaluated	N	24	21	24	23
Fetuses Evaluated	N	156	139	153	142
FIBULA
Bent
Fetal Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	0.7
Litter Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	4.3
Affected fetuses per Litter	MEAN%	0.0	0.0	0.0	0.7
	SD	0.0	0.0	0.0	3.5
METATARSAL
Incomplete ossification of 5th Metatarsal
Fetal Incidence	N	2	0	0	1
	%	1.3	0.0	0.0	0.7
Litter Incidence	N	1	0	0	1
	%	4.2	0.0	0.0	4.3
Affected fetuses per Litter	MEAN%	1.2	0.0	0.0	0.5
	SD	5.8	0.0	0.0	2.3
TOTAL AFFECTED FETUSES
Fetal Incidence	N	70	36	62	100 (a)
	%	44.9	25.9	40.5	70.4
Litter Incidence	N	17	17	17	21
	%	70.8	81.0	70.8	91.3
Affected fetuses per Litter	MEAN%	41.1	26.3	39.3	72.6 (b)
	SD	37.0	25.8	30.5	33.5

(a) Significantly different from control group 1 at 0.001 using Fisher exact  test;

(b) Significantly different from control group 1 at 0.01 using Kruscal-Wallis  test

 

Historical Control Data Summary on Reproductive Toxicity Studies 

Test Facility:

Biological Testing Laboratory (BTL)

Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry

Russian Academy of Sciences (BIBC RAS)

Prospect Nauki 6, Pushchino

Moscow Region 142290

Russia

 

 

HISTORICAL CONTROL SUMMARY DATA OF FORMER IMPLANTATION SITES, NUMBER OF CORPORA LUTEA, IMPLANTATION LOSS INDICES, NUMBER OF FETUSES IN FEMALES EUTHANIZED ON GESTATION DAYS 19-20

Species / Strain:  Rat / Sprague-Dawley
Number of studies: 4 ^(a)	Range of Study Dates:			August 2012 - February 2014
	MEAN	S.D.	N	Min / Max Value
CONTROL – VEHICLE FEMALES
Number of corpora lutea, N per female	14.4	4.0	77	1.0	/	23.0
Number of implantation sites, N per female	13.9	3.2	38	7.0	/	21.0
Pre-implantation loss, N per female	1.9	3.4	38	0.0	/	13.0
Pre-implantation loss (% per female)	10.9	18.5	38	0.0	/	61.9
Live fetuses, N per female	12.7	4.0	77	1.0	/	21.0
Dead fetuses, N per female	0.0	0.0	77	0.0	/	0.0
Post-implantation loss, N per female	0.8	1.1	77	0.0	/	5.0
Post-implantation loss (% per female)	5.8	8.1	77	0.0	/	35.7

^(a) Studies of prenatal toxicity of some clinical drugs carried out in accordance with the methodological guidelines of the Ministry of Health of the Russian Federation

 

HISTORICAL CONTROL SUMMARY DATA OF FETAL BODY WEIGHT ON PRENATAL DAY 20

Species / Strain:  Rat / Sprague-Dawley
Number of studies: 1	Range of Study Dates:			January - February 2014
	MEAN	S.D.	N	Min / Max Value
Mean body weight for male and female fetuses, g	3.91	0.39	258	2.36	/	4.79
Mean fetal body weight per litter, g	3.87	0.35	20	2.75	/	4.43

Note: Separate body weight data for each sex of fetuses on GD20 are not available for historical studies as well as data on AGD and % males in prenatal toxicity studies (before 2019). As historical control data of % males in the litter, the data of studies in accordance with the OECD 422 can be used (see table below).

 

SUMMARY OF HISTORICAL CONTROL DATA ON NUMBER OF PUPS PER LITTER, SEX, AND % MALES

Species / Strain:  Rat / Sprague-Dawley                     Number of Studies: 5 ^(b)
Range of Study Dates:  November 2017 – January 2019
	N	MEAN	SD	MEAN +/- 2 SD		Min	Max
Number of litters	53
Number of pups, N	651
females, N	321
males, N	329
% males, total	50.5
Number of pups per litter		12.3	2.4	7.5	17.1	6.0	19.0
females per litter, N		6.2	2.0	2.2	10.2	2.0	11.0
males per Litter, N		6.0	2.3	1.4	11.1	2.0	11.0

^(b) Screening studies of repeated dose oral toxicity with the reproduction/developmental toxicity (OECD TG 422)

 

 

HISTORICAL CONTROL SUMMARY DATA OF FETAL SKELETAL AND VISCERAL MALFORMATIONS ON GESTATION DAY 19

Species / Strain:  Rat / Sprague-Dawley
Number of studies: 3	Range of Study Dates:  August 2012 - February 2014
SKELETAL MALFORMATIONS
Litters Evaluated, N	54
Fetuses Evaluated, N	461
Fetal incidence, N	0
Fetal incidence, %	0.0
Litter incidence, N	0
Litter incidence, %	0.0
VISCERAL MALFORMATIONS
Litters Evaluated, N	57
Fetuses Evaluated, N	260
Fetal incidence, N	0
Fetal incidence, %	0.0
Litter incidence, N	0
Litter incidence, %	0.0

 

Note: Historical control data on skeletal, cartilage, and visceral variations for fetuses on gestation day 20 are not available before December 2019.

 

 

 

 

 

 

Prepared by Oksana Khokhlova,

Ph.D., senior scientist of BTL BIBC RAS, 04.05.2020

 

 

 

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect-level (NOAEL) for maternal
and developmental toxicity (post-implantation loss) of 1,2,3-Benzotriazole was considered to be 36 mg/kg bw/day.
The no-observed-adverse-effect-level (NOAEL) for embryo-fetal developmental toxicity of 1,2,3-Benzotriazole was considered to be 120 mg/kg bw/day

Executive summary:

Objective

The objective of this prenatal developmental toxicity study was to evaluate the potential effects of the test

item, 1,2,3-Benzotriazole (CAS No. 95-14-7), on pregnancy and on embryo-fetal development in rats

following daily oral (gavage) administration at doses 36, 120 and 330 mg/kg body weight/day from

implantation to the day prior to scheduled caesarean section (day 5 to day 19 post-mating inclusive).

Design

Results from the separate dose-range-finding study as well as published toxicity data of 1,2,3-Benzotriazole

and data from a structurally closely related substance (Methyl-1H-benzotriazole, CAS No. 29385-43-1) were

considered for dose selection.

The test item, 1,2,3-Benzotriazole, in the vehicle (Kollisolv® PEG E 400) was administered by gavage once

daily to Sprague-Dawley female rats from day 5 to day 19 post-mating (inclusive). Three groups received

the test item at dose-levels of 36, 120 or 330 mg/kg bw/day. A concurrent vehicle control group received the

vehicle (Kollisolv® PEG E 400) on a comparable regiment and in the same volume of 3 mL/kg bw. Each

group consists of 24-26 females with confirmed mating.

All females were observed twice daily for mortality and morbidity and for signs of toxicity following dose

administration. Body weights and food consumption are recorded at three-day intervals. On day 20 post-

mating, the dams were sacrificed and subjected to a macroscopic examination and enumeration of corpora

lutea. The weight of the thyroid gland, histopathological assessment of the thyroid gland, and assay of

serum concentration of thyroxin (T4), triiodothyronine (T3), and thyroid stimulating hormone (TSH) were

done in dams to observe pathological changes in thyroid function. Gravid uteri were weighed, and uteri

content are examined to record implantation sites, early and late resorptions, dead, and live fetuses. The

fetuses were weighed, sexed with measurement of anogenital distance (AGD), and submitted to external

examination. Approximately half of the fetuses from each litter were subjected to a detailed examination of

soft tissue by serial sectioning after fixation in Bouin’s solution while the other half underwent detailed

skeletal examination following staining of bone with alizarin red and cartilage with alcian blue.

The fetal findings were described according to the harmonized terminology of the International Federation of

Teratology Societies (IFTS) and classified as malformations or variations. Fetal incidence, litter incidence,

and affected fetuses per litter were calculated for external, visceral, and skeletal alterations. The

reproductive tract was examined with particular attention, and external sex was compared with internal

(gonadal) sex in all fetuses. In addition, male fetuses were examined for undescended testes.  

Results

There was no morbidity and mortality of females caused by the test item administration. The test item

related clinical findings were observed in the 330 mg/kg bw/day dose group after dosing and were as

follows: hypokinesia, staggering gait or flatness as a more severe behavior disorder, excessive vocalization,

chromodacryorrhea, ptyalism, nasal discharge. Clinical observations were episodic with the recovery of

animals within approximately 1.5 – 3 hours after dosing. The higher frequency of clinical signs was noted

after first dosing (gestation day 5), as well as at the end of the administration period (gestation days 14, 15,

17, and 19). Over the entire period of the test item administration, the total number of females with clinical

findings in the 330 mg/kg bw/day dose group was 14 out of 26 examined (p < 0.001, Fisher Exact test). The

body weight of pregnant females administered with the dose of 330 mg/kg bw/day was slightly decreased

compared to the control vehicle group at the gestation day 20 (by 5.0%, p < 0.05), and the body weight gain

was statistically reduced (p < 0.01) the entire period of administration compared with the control group as

well as lower doses. The reduced body weight gain of pregnant females in the 330 mg/kg bw/day dose

group was associated with a smaller weight of the pregnant uterus (by 16 % compared to the control group,

p < 0.05). The final maternal body weight without a gravid uterus did not statistically differ from the body

weight of control females. Mean food consumption in the 36, 120 and 330 mg/kg bw/day group was similar

to that in the vehicle control group during all study days.

During necropsy, there were no gross findings related to the test item administration. The mean number of

corpora lutea and pre-implantation loss did not statistically differ in the test item treated groups compared to

the control vehicle group. The mean number of implantation sites was approximately the same among

groups.  

The test item did not affect the observed thyroid function of pregnant females. The absolute and relative

thyroid weight was not significantly changed in all dose test item treated group. Test item related C-cell

hyperplasia was observed in 3 of 24 females from 120 mg/kg bw/day dose group, and in 4 of 23 females

from 330 mg/kg bw/day groups (p < 0.05, Fisher Exact test). This histological alteration was of minimum

grade and can be associated with the calcitonin exchange (as speculation). Although there is no data on the

serum level of calcitonin and calcium in pregnant females treated with benzotriazole, C-cell hyperplasia may

be considered potentially adverse. The level of thyroid hormones T3, T4, and TSH in the serum of pregnant

females was not significantly changed in the test item treated groups.  

The relative post-implantation loss was increased in the 120 and 330 mg/kg bw/day dose groups compared

to the control vehicle group (p < 0.01 and p < 0.05, respectively). The increase of post-implantation loss in

these groups was associated with the increase in the absolute and percentage values of early resorptions.

Also, the total number of females with post-implantation loss was increased (non-significantly) in the 120

and 330 mg/kg bw/day dose groups. The mean number of fetuses per female was slightly decreased in the

330 mg/kg bw/day dose group (by 6.2 % compared to the control group), but this change was not

significant.

The body weight of male and female fetuses, as well as mean fetuses weight of litter, was significantly

decreased in the 330 mg/kg bw/day dose group (respectively, by 11.2 %, 11.8 %, and 11.5 %). The

absolute and normalized anogenital distance was not changed in male fetuses; however, for female fetuses,

the normalized anogenital distance was statistically increased in the 330 mg/kg bw/day dose group. The

fetal sex ratio in all dose treated groups was not significantly changed compared to the control vehicle

group.

In the 36 mg/kg bw/day dose group, there were no fetuses with external observations, soft tissue, and

skeletal and cartilage alterations recognized as malformations; there were no variations or other alterations

which are considered as test item related.    

In the 120 mg/kg bw/day dose group, three fetuses from one litter were with inward rotated hindlimbs during

an external examination. No skeletal and visceral malformations were observed for these fetuses; however,

the altered ossifications were noted for two of them. Besides, these fetuses had a domed head as an

external observation with unknown importance, and the dilated ventricles of the brain were noted for one of

them. The fetal and litter incidence of dilated brain ventricles (fourth, third, lateral and ventricle) was

statistically increased in the 120 mg/kg bw/day dose group compared to the control group (p < 0.01 for

ventricle) as well as the incidence of the dilated renal pelvis (p < 0.01). These findings were not dose-

dependent. In the 330 mg/kg bw/day dose group, the dilation of the fourth brain ventricle was found in 4

fetuses from 2 litters, and the dilation of the renal pelvis was found in 1 fetus, which was not significantly

higher compared to the control group. So, in the 120 mg/kg bw/day dose group, the dilated brain ventricles

and dilated renal pelvis as soft tissue alterations of unknown importance are considered to be with unclear

relation to the test item administrations. In 120 mg/kg bw/day dose group, there was no significant increase

in the fetal and litter incidence of skeletal and cartilage observations.

In the 330 mg/kg bw/day dose group, one female fetus had following external malformations: inward rotated

hindlimbs, absent of some digits on the left hindlimb, thread-like tail, and absent of anus confirmed during

evisceration. This fetus also had a large abdomen, probably as a result of the absence of the anus; no other

malformations of soft tissues were found during evisceration. The external malformations in the fetus were

associated with the skeletal malformations: the absence of vertebrae after the thoracic T4, and the presence

of only seven ribs on the right and left, of which the 5th, 6th, and 7th pairs were unossified, and unilateral

absence of hindpaw phalanges. Despite the uniqueness of this finding, it considered being test item-related.

In this group, the increase in the incidence of altered fetal ossification was observed. The statistical

significant increase in incidence compared to the control vehicle group was noted for incomplete ossification

of 2nd - 4th sternebra (fetal incidence, p < 0.05), unossification of 6th sternebra (fetal and litter incidence, p

< 0.05), unossification in 5th metacarpal (fetal and litter incidence, p < 0.001 and p < 0.01) or its incomplete

ossification (fetal incidence, p < 0.05). The total affected fetuses with alteration in ossification was

statistically increased for sternebra (fetal incidence, and mean of fetus per litter, p < 0.01), for metacarpal

(fetal and litter incidence, and mean of fetus per litter, p < 0.01), and for total variations (fetal incidence, p <

0.001, and mean of fetus per litter, p < 0.01). Moreover, in the 330 mg/kg bw/day dose group, the statistical

increase in the incidence of soft tissue variations was observed for the liver (discoloration - pale, spotted),

kidney (discolored due to the hemorrhage), and uterus (thin uterine horns). The percentage increase was

observed for the fetal incidence (liver and kidney), as well as for the litter incidence (kidney, uterus, and liver

(non-significant)). The slight non-statistical dose-dependence was noted for these findings, so, they are

considered as test item related. It should be noted that thin uterine horns are regarded as of unknown

importance and may be due to the small fetuses in the high dose group, recorded during an external

examination.

The fetal and litter incidence of testis malposition was the same in the test item treated and control groups.

In most fetuses, this observation was expressed as a unilateral undescended testis at half the distance from

the kidney.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

120 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

only one study available for an analogue

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The performed screening test for Benzotriazole shows no adverse effects up to 200 mg/kg bw/day, a NOAEL of 200 mg/kg bw/day can be derived. The read across from Benzotriazole to Sodium Benzotriazole is feasible. The results of the screening test are of limited use for the characterisation of this hazard as stated in the OECD Guideline: "This test does not provide complete information on all aspects of reproduction and development. In particular, it offers only limited means of detecting post-natal manifestations of prenatal exposure, or effects that may be induced during post-natal exposure."

For the risk characterisation, the NOAEL of 36 mg/kg bw/day from pre-natal developmental study was used.

Justification for selection of Effect on developmental toxicity: via oral route:
A well performed study according to OECD Guideline 414 for an analogue is chosen.

Justification for classification or non-classification

The information on toxicity to reproduction are conclusive but not sufficient for a classification according Regulation (EC) No. 1272/2008.